Molecular Variation and Distribution of Anopheles fluviatilis (Diptera: Culicidae) Complex in Iran

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.     

DNA Barcodes indicate members of the Anopheles fluviatilis (Diptera: Culicidae) species complex to be conspecific in India

Anopheles fluviatilis, a major vector of malaria in India has been described as a complex of three sibling species members, named as S, T and U, based on variations in chromosomal inversions. Also, ribosomal DNA markers (repetitive Internal Transcribed Spacer 2 (ITS2) and 28S D3 region) were described to differentiate these three sibling species members. However, controversies prevail on the genetic isolation status of these cryptic species. Hence, we evaluated this taxonomic incongruence employing DNA barcoding, the well established methodology for species identification, using 60 An. fluviatilis sensu lato specimens, collected from two malaria endemic eastern states of India. These specimens were also subjected to sibling species characterization by ITS2 and D3 DNA markers. The former marker identified 31 specimens among these as An. fluviatilis S and 21 as An. fluviatilis T. Eight specimens amplified DNA fragments specific for both S and T. The D3 marker characterized 39 specimens belonging to species S and 21 to species T. Neither marker identified species U. Neighbor Joining analysis of mitochondrial cytochrome c oxidase gene 1 sequences (the DNA barcode) categorized all the 60 specimens into a single operational taxonomic unit, their Kimura 2 parameter (K2P) genetic variability being only 0.8%. The genetic differentiation (F_ST) and gene flow (N_m) estimates were 0.00799 and 62.07, respectively, indicating these two ‘species’ (S & T) as genetically con‐specific intermixing populations with negligible genetic differentiation. Earlier investigations have refuted the existence of species U. Also, this study demonstrated that An. fluviatilis and the closely related An. minimus could be taxonomically differentiated by the DNA Barcode approach (K2P = 5.0%).     

Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Molecular phylogeny ofSalicaceae and closely relatedFlacourtiaceae: Evidence from 5.8 S, ITS 1 and ITS 2 of the rDNA

A ribosomal DNA region, including the entire 5.8S RNA gene and the internal transcribed spacers ITS 1 and ITS 2, was used for studying the phylogeny ofSalicaceae and the relationship betweenSalicaceae andFlacourtiaceae. The length of the ITS regions withinSalicaceae andFlacourtiaceae was similar to that found in other angiosperms. The GC content of both ITS regions was high, varying 62.7-72.2%. The most parsimonious tree clusters the wind-pollinatedChosenia bracteosa among theSalix species, suggesting that it should be included in the genusSalix. The grouping withinSalix leaves subg.Salix as paraphyletic, for which reason the subgeneric division is questionable.Populus was monophyletic and formed a sister group toSalix. The interspecific variation of the ITS sequences was very small inSalicaceae, which is in contradiction to the age of the group according to the evidence from fossil data.Idesia polycarpa fromFlacourtiaceae shows great sequence similarity withSalicaceae, but the analysis of 5.8S rDNA supports monophyly of the four species ofFlacourtiaceae sampled for this study.     

Molecular phylogeny of species in the generaAmylostereum andEchinodontium

Analyses of DNA sequences from the internal transcribed spacer (ITS) region of the nuclear rDNA and from a portion of a manganese-dependent peroxidase gene were used to assess the species inAmylostereum, including isolates from the mycangia of horntails, decay, and basidiomes. Four species are recognized:A. areolatum, A. chailletii, A. laevigatum, andA. ferreum. An unidentifiedAmylosterum isolate from the mycangium ofXoanon matsumurae had an ITS sequence identical to that ofA. areolatum. Another unidentifiedAmylostereum isolate from the mycangium ofSirex areolatus was nearA. laevigatum, which appears to be the mycangial symbiont for those horntails attacking cedar-like trees. The other horntail isolates, primarily from Pinaceae, proved to be eitherA. areolatum orA. chailletii. The DNA sequences ofEchinodontium tinctorium, E. tsugicola andE. japonicum were similar to those of theAmylostereum species, andAmylostereum species are now recognized as members of the family Echinodontiaceae rather than the family Stereaceae.Echinodontium taxodii was found to be distinct from the Echinodontiacaea andStereum, andE. taxodii is recognized as aLaurilia species.     

An allele-specific polymerase chain reaction assay for the differentiation of members of the<Emphasis Type="Italic">Anopheles culicifacies</Emphasis> complex

Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminatesAn. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally-identified specimens ofAn. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.     

Molecular characterization and identification ofSaprolegnia by restriction analysis of genes coding for ribosomal RNA

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species ofSaprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except forS. hypogyna, S. delica, S. lapponica, andS. mixta. The rDNA data indicate thatS. lapponica andS. mixta are conspecific withS. ferax, whereas there is no support for the proposed synonymies ofS. diclina withS. delica and ofS. mixta withS. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in theS. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains ofS. parasitica originating from the U.K. and Japan.HhaI andBsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonucleaseBstUI was diagnostic forS. parasitica, generating identical fingerprints for all strains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved inS. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed thatS. diclina andS. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification ofSaprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.     

Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the Myzomyia Series from Southeast Asia

Abstract. Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Dönitz, An. pampanai Büttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA‐based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.     

Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review,distribution and malaria vector status

Abstract. Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single‐strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990 ) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984 ) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type‐locality: Pokfulam, Hong Kong). The so‐called subspecies x of An. minimus (sensu Baba, 1950 ) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.     

Genome characterization and relationships between two species of the genusLobelia (Campanulaceae) determined by repeated DNA sequences

Three repeated DNA sequences (rDNA 5S, 18S-5.8S-26S and telomeric repeats) were localised in the genomes ofLobelia brasiliensis andL. imperialis var.kanitzii (subg.Tupa), both with 2n = 28, by fluorescence in situ hybridization (FISH). The results were used to analyse the genomic relationship between the species. With probe pTa71, the karyotypes of these species showed only one NOR site. Probe pTa794, which contains 5S rDNA, demonstrated differences between the species. Telomeric sequences, studied with probe pLT11, were not detected in ectopic sites, but different telomeres showed signals of varying intensity. Based on the results obtained, considerations are made on karyotype evolution inLobelia.     

RAPD patterns,nrDNA ITS sequences and morphological patterns inSilene sectionSedoideae (Caryophyllaceae)

Hierarchical patterns inSilene sect.Sedoideae were investigated using random amplified polymorphic DNA (RAPD), nucleotide sequences of the internal transcribed spacer (ITS) regions of the 18S–28S nuclear ribosomal DNA, and discrete morphological characters. All data sets firmly supported the species recognized. The RAPD data offered the best resolution at the intraspecific level, supporting the current intraspecific classifications ofS. sedoides andS. integripetala. The ITS sequences and the morphological data gave poor resolution within species, and the three data sets disagreed about the relationships among species. The signal from the RAPD data was strongest and remained when the total data set was analysed. The three data sets all support an amphiploid origin ofS. aegaea, with the strongest evidence from the ITS sequences. Incongruences among data sets as well as merits and shortcomings of each are discussed. The robustness of the results can be evaluated using perturbations of data, i.e., bootstrap and jackknife of taxa and characters. These methods should not be taken as methods of statistical inference at the taxonomic level, because unbiased sampling appears impossible. RAPD data, however, come close to being suitable for statistical estimation of hierarchies at the genome level, but several methodological problems have to be solved.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Protential use of PCR-amplified ribosomal intergenic sequences for differentiation of varieties and species ofGossypium cotton

Our purpose was to develop a new approach to the identification ofGossypium cotton varieties and species based on polymerase chain reaction (PCR). Species-specific distinctions within the genusGossypium have been detected by the amplification of ribosomal genes, namely theRrn18-Rrn25 internal transcribed spacer (ITS) regions that had sequence differences. Using the primers to the 3′-end ofRrn18 adjacent to ITS1 and the 5′-end ofRrn25 adjacent to ITS2 from tomato, we have obtained amplified fragments of two cotton species,G. barbadense andG. herbaceum. Interspecies distinctions have been revealed by the restriction assay of these amplification products. The restriction patterns are distinguished not only by number but by location and intensity of the bands. Our results illustrate the effective use of differences in ribosomal intergenic sequences for the differentiation of varieties and species ofGossypium.     

Xenocylindrocladium guianense andX. subverticillatum, two new species of hyphomycetes from plant debris in the tropics

Two new species of hyphomycetes,Xenocylindrocladium guianense andX. subverticillatum, are described from plant debris collected in French Guiana and Singapore, respectively. The genusXenocylindrocladium has thus far been known from one species,X. serpens, which was described from plant debris collected in Ecuador. The two new taxa are compared with and distinguished fromX. serpens based on morphology, cultural characteristics and phylogenetic analysis of DNA sequence data of the 5.8S rDNA with flanking ITS1 and ITS2 regions and the 5′ end of the β-tubulin gene. These species are also compared with other closely related hypocrealean taxa. Present collection data suggest that species ofXenocylindrocladium could be restricted to the tropics.     

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.     

Phylogenetic relationships ofPythium species based on ITS and 5.8S sequences of the ribosomal DNA

The sequences of ITS regions in 30 species and two groups of the genusPythium were resolved. In the phylogenetic trees, the species were generally divided into two clusters, referred to here as the F and S groups. The species in the two groups correspond in terms of their sporangial morphology, with the F group being filamentous/lobulate and the S group being spherical. Genetic divergence within the F group was lower than that within the S group. Other morphological characteristics such as oogonial structure and sexual nature appeared to be unrelated to the groupings in these trees. An alignment analysis revealed common sequences to all the species and arrangements specific to each F or S group. It was found that the ITS region was a good target in designing species-specific primers for the identification and detection ofPythium species. In the tree based on 5.8S rDNA sequences, oomycetes are distantly related to other fungi but separated from algae in Chromista.     

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid <Emphasis Type="Italic">Aegilops</Emphasis> (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 193–197.Original Russian Text Copyright © 2005 by Goryunova, Chikida, Gori, Kochieva.     

Phylogeny of anopheline (Diptera: Culicidae) species in southern Africa,based on nuclear and mitochondrial genes

A phylogeny of anthropophilic and zoophilic anopheline mosquito species was constructed, using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The ITS2 alignment, typically difficult due to its noncoding nature and large size variations, was aided by using predicted secondary structure, making this phylogenetically useful gene more amenable to investigation. This phylogeny is unique in explicitly including zoophilic, non‐vector anopheline species in order to illustrate their relationships to malaria vectors. Two new, cryptic species, Anopheles funestus‐like and Anopheles rivulorum‐like, were found to be present in Zambia for the first time. Sequences from the D3 region of the 28S rDNA suggest that the Zambian An. funestus‐like may be a hybrid or geographical variant of An. funestus‐like, previously reported in Malawi. This is the first report of An. rivulorum‐like sympatric with An. rivulorum (Leeson), suggesting that these are separate species rather than geographic variants.     

Molecular phylogenetic relationships inAveneae (Poaceae) species and other grasses as inferred from ITS1 and ITS2 rDNA sequences

A phylogenetic analysis was conducted on sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA in 23 species ofAveneae (Poaceae subfam.Pooideaae). These sequences ofHelictotrichon spp.,Arrhenatherum elatius, Avena spp.,Trisetum spp.,Koeleria spp.,Holcus lanatus, Alopecurus vaginatus together with published ITS sequences of furtherAveneae, Poeae, Triticeae, andBromeae were analysed by the neighbor-joining distance method to assess the molecular phylogenetic relationship in perennial and annualAveneae. The results suggest unexpectedly close affinities of the agronomically important genusAvena to comparatively small-flowered taxa ofAveneae. GenusArrhenatherum and small-flowered subgenera ofHelictotrichon are close extant relatives. The large genusHelictotrichon is para- if not polyphyletic, only its subgenera are monophyletic.Trisetum is clearly separated fromHelictotrichon and forms together withKoeleria and perhaps others a monophyletic lineage which is characterised by a conspicuous 9-bp deletion in ITS1. The impact of the ITS data on the delineation of some genera and subtribes ofAveneae and on the recognition of their biogeographical and ecological patterns is outlined.