Ribosomal Internal Transcribed Spacer Research Articles

Phylogenetic analysis of eastern Asian and eastern North American disjunct Lespedeza (Fabaceae) inferred from nuclear ribosomal ITS and plastid region sequences

Lespedeza (tribe Desmodieae, Fabaceae) follows a disjunct distribution in eastern Asia and eastern North America. Phylogenetic relationships among its species and related taxa were inferred from nuclear ribosomal internal transcribed spacer (ITS) and plastid sequences (trnH-psbA, psbK-psbI, trnK-matK and rpoC1). We examined 35 species of Lespedeza, two of Kummerowia and one of Campylotropis, the sole constituents of the Lespedeza group. An analysis of these data revealed that the genus Campylotropis is sister to the other two genera. However, we were unable to resolve the relationships between Kummerowia and Lespedeza in the strict consensus trees of parsimony analyses based on plastid and combined DNA data. In the genus Lespedeza, the Old World subgenus Macrolespedeza is monophyletic, whereas the transcontinental subgenus Lespedeza is paraphyletic. Monophyly of eastern Asian species and of North American species is strongly supported. Although inconsistent with the traditional classification, this phylogenetic finding is consistent with seedling morphology. Three subgroups recognized in subgenus Macrolespedeza were unresolved in our phylogenetic trees. An incongruence length difference (ILD) test indicated that the two partitions (nuclear ITS and plastid sequences) were significantly incongruent, perhaps because of hybridization between species in Lespedeza. Most of the primary clades of tribe Desmodieae are Asian, implying that the relatively few New World ones, such as those in Lespedeza, are more recently derived from Asia. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 221–235.

Identification of insect cell lines and cell-line cross-contaminations by nuclear ribosomal ITS sequences

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR-RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR-based method used three species-specific primer sets, Ly-ITS1/Ly-ITS2, ITS1-1/Ld-ITS1 and Sf9-F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell-line cross-contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell-line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.

First Report of Leaf Spot on Horseweed (Conyza canadensis) Caused by Septoria erigerontis in Turkey.

Horseweed (Conyza canadensis (L).Cronq., Asteraceae) is an invasive exotic weed in Turkey and a problematic native weed in the United States where glyphosate-resistant populations of the weed have developed (2). These characteristics make horseweed a target for biological control efforts. In September 2009, small, brown leaf spots were observed on leaves of C. canadensis in Taflan, Turkey (41°25.398'N, 36°08.352'E). Globose, dark-walled pycnidia were also observed in brown spots on leaves. Diseased tissue was surface disinfested and placed on moist filter paper in petri plates. A fungus designated 09-Y-TR1 was isolated from the diseased leaves. Single-spore isolations were grown on potato dextrose agar (PDA). Cultures on PDA formed dark green-to-black colonies. Pycnidia matured after 3 to 4 weeks when plates were incubated at 23°C with a 12-h photoperiod (black light and cool white fluorescent light). Pycnidia were separate, immersed, and dark brown with a single apical ostiole. Matured conidia were one to three septate, filiform, straight to slightly curved, rounded at the apex, smooth walled, hyaline, and 22 to 40 × 1.4 to 2.5 μm. Morphology was consistent with Septoria erigerontis Peck (3). Comparison of the internal transcribed spacer (ITS) 1 and 2 sequence with available sequences of vouchered S. erigerontis specimens (GenBank EF535638.1, AY489273.1; KACC 42355, CBS 109094) showed 447 of 450 and 446 of 450 identities, respectively. Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2, including 5.8S rDNA) were deposited in GenBank (GU952666). For pathogenicity tests conidia were harvested from 3-week-old cultures grown on PDA, by brushing the surface of the colonies with a small paint brush, suspended in sterile distilled water, and filtered through cheese cloth. Conidia were then diluted in sterile distilled water plus 0.1% polysorbate 20 to a concentration of 5 × 106 conidia/ml. Stems and leaves of seven 5-month-old seedlings were spray inoculated with 10 ml of this aqueous suspension per plant. Inoculated plants and three noninoculated plants were placed in a dew chamber at 23°C in darkness and continuous dew, and after 48 h, plants were moved to a greenhouse bench. Symptoms were observed 2 days after inoculation. Disease severity was evaluated 2 weeks after inoculation by a rating system with a scale of 0 to 6 based on percentage of plant tissue necrosis, in which 0 = no symptoms, 1 = 1 to 5%, 2 = 6 to 25%, 3 = 26 to 75%, 4 = 76 to 95%, 5 = >95%, and 6 = dead plant. The average disease rating on inoculated plants was 3.55. No disease was observed on noninoculated plants. S. erigerontis was reisolated from all inoculated plants. To our knowledge, this is the first report of leaf spot on horseweed caused by S. erigerontis in Turkey where the fungus may have potential as a classical biological control agent. S. erigerontis has also been reported on C. canadensis in Korea and Portugal (1). In the United States, S. erigerontis has been reported on horseweed in several states (1) and these isolates may have potential as biological control agents of horseweed, particularly glyphosate-resistant horseweed, in the United States.

Closing the gaps: phylogenetic relationships in the Brassicaceae based on DNA sequence data of nuclear ribosomal ITS region

Sequence data from the nuclear encoded ribosomal internal transcribed spacer (ITS) region were used to determine monophyly of tribes, tribal limits, and tribal relationships of 96 so far unassigned or tentatively assigned genera (represented by 101 taxa/accessions) within the Brassicaceae. Maximum-parsimony and maximum-likelihood analyses of 185 ITS Brassicaceae sequences, which also included representatives of each of the 34 currently recognized tribes, supported the separate phylogenetic distinctness of these tribes and permitted the tribal assignment of all but 12 of the unassigned genera into tribal clades. The data support the recognition of eight new, well-resolved, uni- or oligogeneric tribes recognized herein as the Alyssopsideae [96% bootstrap support (BS); including the central and southwestern Asian Alyssopsis and Calymmatium], Asteae (100% BS; including the Mexican Asta), Eudemeae (97% BS; South American Brayopsis, Eudema, and Xerodraba), Kernereae (96% BS; European Kernera and Rhizobotrya), Notothlaspideae (100% BS; New Zealandic Notothlaspi), Oreophytoneae (100% BS; eastern African Oreophyton and southern European Murbeckiella), and Yinshanieae (100% BS; Chinese Yinshania), as well as the moderately supported Microlepidieae (75% BS; Australian Microlepidium and Carinavalva). Furthermore, the results fully support the recent findings that the tribes Schizopetaleae and Thelypodieae ought to be recognized as two distinct tribes instead of a single tribe, as well as provide some support for the re-establishment of the tribe Cremolobeae, bringing the total number to 44 tribes in the family. Nearly 92% (308) of the 336 genera in the family have been assigned to a tribe. The earlier-published Anastaticeae is taken here to replace the Malcolmieae.

Development of ITS sequence based molecular marker to distinguish, Tribulus terrestris L. (Zygophyllaceae) from its adulterants

Tribulus terrestris L. (Zygophyllaceae) is one of the highly traded raw drugs and also used as a stimulative food additive in Europe and USA. While, Ayurvedic Pharmacopoeia of India recognizes T. terrestris as Goksura, Tribulus lanuginosus and T. subramanyamii are also traded by the same name raising issues of quality control . The nuclear ribosomal RNA genes and ITS (internal transcribed spacer) sequence were used to develop species-specific DNA markers. The species-specific markers efficiently amplified 295 bp for T. terrestris (TT1F and TT1R), 300 bp for T. lanuginosus (TL1F and TL1R) and 214 bp for T. subramanyamii (TS1F and TS1R). These DNA markers can be used to distinguish T. terrestris from its adulterants.

First Report of Leaf Anthracnose Caused by Phomopsis convolvuli on Field Bindweed in Turkey.

Field bindweed (Convolvulus arvensis L.; Convolvulaceae) is a troublesome perennial weed found among many important crops in the world (1). In May of 2007, dying field bindweed plants were found along the edge of a wheat (Triticum aestivum L.) field between Bafra and Taflan, Turkey (41°34.395'N, 35°52.215'E). Lesions on leaves were irregular and variable in size and dark black with green margins. Severely diseased leaves were wilted or dead. Fruiting bodies were not evident on field-collected material. Diseased tissue was surface disinfested and placed on moist filter paper in petri plates. Numerous pycnidia with alpha conidia were observed after 2 weeks. A fungus, designated 24-6, was isolated from the diseased leaves. Cultures on potato dextrose agar (PDA) were floccose with white mycelia and small black stromata. Alpha conidia from pycnidia on inoculated plants were biguttulate, one celled, hyaline, oblong to ellipsoid, and 7.0 to 12.8 × 3.0 to 5.5 μm (mean 10.0 × 3.9 μm). Neither beta conidia nor the teleomorph, Diaporthe sp., were observed on diseased tissue or in cultures. Morphology was consistent with that of Phomopsis convolvuli Ormeno-Nunez, Reeleder & A.K. Watson (2). Alpha conidia were harvested from 12-day-old cultures grown on PDA by brushing the surface of the colonies with a small paint brush, suspending the conidia in sterile distilled water, and filtering through cheesecloth. The conidia were then resuspended in sterile distilled water plus 0.1% polysorbate 20 to arrive at a concentration of 107 conidia/ml. Stems and leaves of seven plants at the 3- to 5-leaf stage were spray inoculated with 10 ml per plant of this aqueous suspension. Inoculated plants and two noninoculated plants were placed in a dew chamber at 24°C in darkness and continuous dew. After 48 h, plants from the dew chamber were moved to a greenhouse bench. Disease severity was evaluated 1 week after inoculation with a rating system based on a scale from 0 to 4, in which 0 = no symptoms, 1 = 1 to 25% necrosis, 2 = 26 to 50% necrosis, 3 = 51 to 75% necrosis, and 4 = 76 to 100% necrosis (2). The average disease rating on inoculated plants was 3.75. No disease was observed on noninoculated plants. P. convolvuli was reisolated from all inoculated plants. Comparison of the internal transcribed spacer (ITS) 1 and 2 sequences with available sequences of a vouchered P. convolvuli specimen (GenBank Nos. U11363, U11417; BPI 748009, FAU649) showed 192 of 193 and 176 of 179 identities, respectively, for the two regions. Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2, including 5.8S rDNA) were deposited in GenBank (Accession No. FJ710810), and a voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 878927). To our knowledge, this is the second report in the world of leaf anthracnose on field bindweed caused by P. convolvuli. The first report was from Canada (3) of an isolate that was later patented for biological control of C. arvensis (4).

First Report of Stem Canker of Salsola tragus Caused by Diaporthe eres in Russia.

Salsola tragus L. (Russian thistle) is a problematic invasive weed in the western United States and a target of biological control efforts. In September of 2007, dying S. tragus plants were found along the Azov Sea at Chushka, Russia. Dying plants had irregular, necrotic, canker-like lesions near the base of the stems and most stems showed girdling and cracking. Stem lesions were dark brown and contained brown pycnidia within and extending along lesion-free sections of the stems and basal portions of leaves. Diseased stems were cut into 3- to 5-mm pieces and disinfested in 70% ethyl alcohol. After drying, stem pieces were placed into petri dishes on the surface of potato glucose agar. Numerous, dark, immersed erumpent pycnidia with a single ostiole were observed in all lesions after 2 to 3 days. Axenic cultures were sent to the Foreign Disease-Weed Science Research Unit, USDA, ARS, Ft. Detrick, MD for testing in quarantine. Conidiophores were simple, cylindrical, and 5 to 25 × 2 μm (mean 12 × 2 μm). Alpha conidia were biguttulate, one-celled, hyaline, nonseptate, ovoid, and 6.3 to 11.5 × 1.3 to 2.9 μm (mean 8.8 × 2.0 μm). Beta conidia were one-celled, filiform, hamate, hyaline, and 11.1 to 24.9 × 0.3 to 2.5 μm (mean 17.7 × 1.2 μm). The isolate was morphologically identified as a species of Phomopsis, the conidial state of Diaporthe (1). The teleomorph was not observed. A comparison with available sequences in GenBank using BLAST found 528 of 529 identities with the internal transcribed spacer (ITS) sequence of an authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514; BPI 748435; CBS 109767). Morphology is consistent with that of Phomopsis oblonga (Desm.) Traverso, the anamorph of D. eres (2). Healthy stems and leaves of 10 30-day-old plants of S. tragus were spray inoculated with an aqueous suspension of conidia (1.0 × 106 alpha conidia/ml plus 0.1% v/v polysorbate 20) harvested from 14-day-old cultures grown on 20% V8 juice agar. Another 10 control plants were sprayed with water and surfactant without conidia. Plants were placed in an environmental chamber at 100% humidity (rh) for 16 h with no lighting at 25°C. After approximately 24 h, plants were transferred to a greenhouse at 20 to 25°C, 30 to 50% rh, and natural light. Stem lesions developed on three inoculated plants after 14 days and another three plants after 21 days. After 70 days, all inoculated plants were diseased, four were dead, and three had more than 75% diseased tissue. No symptoms occurred on control plants. The Phomopsis state was recovered from all diseased plants. This isolate of D. eres is a potential biological control agent of S. tragus in the United States. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 878717). Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2) were deposited in GenBank (Accession No. EU805539). To our knowledge, this is the first report of stem canker on S. tragus caused by D. eres.

Nuclear ribosomal DNA diversity of a cotton pest (Rotylenchulus reniformis) in the United States

The reniform nematode (Rotylenchulus reniformis) has emerged as a major cotton pest in the United States. A recent analysis of over 20 amphimictic populations of this pest from the US and three other countries has shown no sequence variation at the nuclear ribosomal internal transcribed spacer (ITS) despite the region’s usual variability. We investigated this unexpected outcome by amplifying, cloning and sequencing two regions of the nuclear ribosomal DNA (18S, ITS1) to ascertain whether any variation occurred within and among populations of reniform nematodes in Alabama, US. Both the nrITS1 and the relatively conserved 18S region showed a fairly substantial amount of variation among populations. The identity among ITS sequences ranged from 1.00 to 0.86, while sequence identity at the 18S ranged from 1.00 to 0.948. We conclude that variation does exist in these sequences in reniform nematodes, and the earlier report showing no ribosomal ITS variation in this pest might have been caused by preferential amplification of a conserved ITS paralog. Current and future application towards resistance in cotton varieties to this pest requires reliable information on the molecular variability of the nematode in cotton-growing areas. Key words: Ribosomal DNA, ITS, 18S, reniform nematode.

Phylogenetic relationships in the tribe Alysseae (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region of 85 species (131 accessions) were used to determine the tribal limits, monophyly status, and phylogenetic intra-tribal relationships of genera within the tribe Alysseae (Brassicaceae). Both maximum parsimony and maximum likelihood analyses support the recognition of the tribe Alysseae s. str. (12 genera: Alyssoides , Alyssum , Aurinia , Berteroa , Bornmuellera , Clastopus , Clypeola , Degenia , Fibigia , Galitzkya , Hormathophylla , and Physoptychis ). Six well-supported clades were recognized within the Alysseae clade, including two Alyssum clades (one of which includes Clypeola ), an Alyssoides and allies clade (includes Alyssoides , Bornmuellera , Clastopus , Degenia , Fibigia , Hormathophylla , and Physoptychis ), a Berteroa and allies clade (includes Aurinia , Berteroa , and Galitzkya ), a Bornmuellera clade, and a Hormathophylla clade. Morphological and cytological support for these clades is reviewed. The ITS data support the exclusion of the following taxa from the Alysseae, with appropriate tribal assignment given in parentheses: Alyssum klimesii Al-Shehbaz (Camelineae), Asperuginoides (unresolved), Athysanus (Arabideae), Botschantzevia (Arabideae), Didymophysa (unresolved), Farsetia (Malcolmieae), Lobularia (Malcolmieae), and Ptilotrichum (Arabideae). Farsetia and Lobularia are inferred to be monophyletic, and based on molecular and morphological characters they are assigned to Malcolmieae, a recently described tribe.

Nuclear ribosomal DNA diversity of a cotton pest ( Rotylenchulus reniformis ) in the United States

The reniform nematode (Rotylenchulus reniformis) has emerged as a major cotton pest in the United States. A recent analysis of over 20 amphimictic populations of this pest from the US and three other countries has shown no sequence variation at the nuclear ribosomal internal transcribed spacer (ITS) despite the region’s usual variability. We investigated this unexpected outcome by amplifying, cloning and sequencing two regions of the nuclear ribosomal DNA (18S, ITS1) to ascertain whether any variation occurred within and among populations of reniform nematodes in Alabama, US. Both the nrITS1 and the relatively conserved 18S region showed a fairly substantial amount of variation among populations. The identity among ITS sequences ranged from 1.00 to 0.86, while sequence identity at the 18S ranged from 1.00 to 0.948. We conclude that variation does exist in these sequences in reniform nematodes, and the earlier report showing no ribosomal ITS variation in this pest might have been caused by preferential amplification of a conserved ITS paralog. Current and future application towards resistance in cotton varieties to this pest requires reliable information on the molecular variability of the nematode in cotton-growing areas.

Phylogenetic patterns and conservation among North American members of the genus Agalinis (Orobanchaceae).

BackgroundNorth American Agalinis Raf. species represent a taxonomically challenging group and there have been extensive historical revisions at the species, section, and subsection levels of classification. The genus contains many rare species, including the federally listed endangered species Agalinis acuta. In addition to evaluating the degree to which historical classifications at the section and subsection levels are supported by molecular data sampled from 79 individuals representing 29 Agalinis species, we assessed the monophyly of 27 species by sampling multiple individuals representing different populations of those species. Twenty-one of these species are of conservation concern in at least some part of their range.ResultsPhylogenetic relationships estimated using maximum likelihood analyses of seven chloroplast DNA loci (aligned length = 11 076 base pairs (bp) and the nuclear ribosomal DNA ITS (internal transcribed spacer) locus (733 bp); indicated no support for the historically recognized sections except for Section Erectae. Our results suggest that North American members of the genus comprise six major lineages, however we were not able to resolve branching order among many of these lineages. Monophyly of 24 of the 29 sampled species was supported based on significant branch lengths of and high bootstrap support for subtending branches. However, there was no statistical support for the monophyly of A. acuta with respect to Agalinis tenella and Agalinis decemloba. Although most species were supported, deeper relationships among many species remain ambiguous.ConclusionThe North American Agalinis species sampled form a well supported, monophyletic group within the family Orobanchaceae relative to the outgroups sampled. Most hypotheses regarding section- and subsection-level relationships based on morphology were not supported and taxonomic revisions are warranted. Lack of support for monophyly of Agalinis acuta leaves the important question regarding its taxonomic status unanswered. Lack of resolution is potentially due to incomplete lineage sorting of ancestral polymorphisms among recently diverged species; however the gene regions examined did distinguish among almost all other species in the genus. Due to the important policy implications of this finding we are further evaluating the evolutionary distinctiveness of A. acuta using morphological data and loci with higher mutation rates.

Detection of Phytophthora nicotianae and P. palmivora in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.

Prevalence and molecular characteristics of Enterocytozoon bieneusi in cattle in Korea

Enterocytozoon bieneusi is the most common microsporidium associated with AIDS patients. Moreover, its detection in increasing numbers of immunocompetent patients has made it an emerging pathogen. This organism was also identified in a wide range of animals, and the zoonotic potential of human infections is of particular interest. In this study, 538 fecal samples from cattle in Korea were analyzed for the presence of E. bieneusi by PCR. Approximately 15% were found to be positive, with higher rates being detected over the summer months. The internal transcribed spacer (ITS) regions of the rRNA gene of ten E. bieneusi positive samples were amplified using nested PCR and sequenced. Genetic polymorphisms, which were represented by six distinct genotypes (CEbA-CEbF), were found among the E. bieneusi isolates. Five isolates from this study had identical ribosomal ITS to the previously known E. bieneusi genotype ITSs in cattle and other animals. Four isolates were previously unreported but were quite similar to the previously known genotypes of E. bieneusi from cattle and other animals. One isolate was identical to the human E. bieneusi type D, which indicated some E. bieneusi isolates from cattle in the country may be of public health importance. To the best of my knowledge, this is the first report of E. bieneusi study in cattle in Asia.

Relatedness Among Agronomically Important Rusts Based on Mitochondrial Cytochrome b Gene and Ribosomal ITS Sequences

AbstractA fragment of the mitochondrial cytochrome b (cyt b) gene of 13 agronomically important plant pathogenic Basidiomycetes was sequenced, including several Puccinia spp., Uromyces appendiculatus, Phakopsora pachyrhizi, Hemileia vastatrix and Rhizoctonia solani. The deduced amino acid sequences (residues 142–266) were used to study the relatedness of these pathogens as compared to other species of the Basidiomycetes, Ascomycetes and Oomycetes. The relatedness was also studied at nuclear level using the Internal Transcribed Spacers (ITS) in the ribosomal DNA. Phylogenic trees were constructed with the maximum parsimony (MP) and the neighbour‐joining (NJ) methods. On the basis of both cytochrome b and ITS sequences, the Puccinia species pathogenic to graminaceous crop plants, such as Puccinia recondita f. sp. tritici, Puccinia graminis f. sp. tritici, Puccinia striiformis f. sp. tritici, Puccinia coronata f. sp. avenae, Puccinia hordei, P. recondita f. sp. secalis and Puccinia sorghi, together with Puccinia horiana from Chrysanthemum, were very closely related to each other, whereas Puccinia arachidis (from peanut) was closely related to U. appendiculatus (from beans) but more distant from the other Puccinia species. Both rusts on soybean (P. pachyrhizi) and coffee (H. vastatrix) were outside the Puccinia cluster. All rusts were separated from other Basidiomycetes such as R. solani and the strobilurin producing species Strobilurus tenacellus and Mycena galopoda. Our results demonstrate that the amino acid sequence of the mitochondrial cytochrome b is a valid tool to study phylogenic relatedness among plant pathogenic Basidiomycetes and supports taxonomic grouping based on morphological structures and host specificity. Because of their high variability, ITS sequences were able to discriminate Puccinia species, which were identical on the basis of the cytochrome b amino acid sequence. Thus, ITS sequences could better show differences among species or within a species, whereas cytochrome b is more suitable than ITS for phyologenic inference at family or genus level. In addition, the sequence data obtained during this study represent essential information for easy isolation of the cyt b gene and detection of point mutations conferring resistance to Qo Inhibitor fungicides that eventually may evolve.

A ‘dirty’ business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.

Refugia, dispersal and divergence in a forest archipelago: a study of Streptocarpus in eastern South Africa

We describe a scenario of plant speciation across a relict forest archipelago in South Africa involving Pleistocene habitat expansion-contraction cycles, dispersal and adaptation to lower temperatures. This is the first population level study using molecular data in South African forests and has significant implications for conservation efforts in this area. Populations of the mesophytic forest floor herbs Streptocarpus primulifolius sensu lato and Streptocarpus rexii were sampled throughout their range in the naturally fragmented forests of eastern South Africa in order to investigate population genetic and phylogenetic patterns within the species complex, using nuclear microsatellites, nuclear ribosomal ITS (internal transcribed spacer) sequences and chloroplast genome sequences. S. primulifolius harbours high levels of genetic diversity at both the nuclear (mean HE = 0.50) and the chloroplast level (each population fixed for a unique haplotype). This is consistent with populations of these coastal species being Pleistocene relicts. In contrast, populations of S. rexii in cooler habitats at higher altitudes and lower latitudes harbour little or no nuclear genetic diversity (mean HE = 0.09) and most share a common chloroplast haplotype. The split of S. rexii from populations intermediate between the two species (S. cf. primulifolius) occurred between 0 and 0.44 million years ago according to the calibrated ITS phylogeny of the taxa. The low genetic diversity and homogeneity of S. rexii is congruent with this species having reached its current range during the Holocene. We found no evidence of monophyly of any of the taxa in this study, which we consider a consequence of recent evolution in a fragmented habitat.

DNA sequencing and genetic diversity of the 18S–26S nuclear ribosomal internal transcribed spacers (ITS) in nine Antarctic moss species

We have sequenced the 18S–26S nuclear ribosomal DNA ITS region from the genome of nine different moss species from the Ross Sea region of Antarctica. This relatively quick and simple technique enables these species to be readily distinguished, facilitating their taxonomic identification. Only a single moss shoot is required, and for identification of these bryophytes it is only necessary to determine a few hundred nucleotides of the DNA sequence in a single sequencing reaction. Several previously unidentified Antarctic moss specimens were readily characterized by comparison with ITS sequences of known moss species. The relationships between species and locations previously detected by the RAPD (Random Amplified Polymorphic DNA) technique were confirmed by DNA sequencing, demonstrating that the two techniques can be complementary for molecular analysis of the ecology of mosses in Antarctica.

(293) Single Nucleotide Polymorphism in matK and Ribosomal ITS of Astilbe

The genus Astilbe (Saxifragaceae) comprises about 13 species and is ranked consistently among the top 10 landscape perennials. Through extensive hybridization, selection and marketing, the lineage of many Astilbehas been lost. Subdioecious Astilbebiternatais the only species in the genus native to North America while other members of the genus are endemic to Asia and monoecious. Due to the unusual geographic distribution of the species and the variation in floral development among them, development of genetic markers using single nucleotide polymorphisms (SNPs) would confirm phylogenetic relationships and establish lineage within the genus. Astilbespecies, hybrids, and cultivars were obtained from plant nurseries and botanical gardens across the country. To elucidate relationships among the genus, we conducted phylogenetic analysis of DNA sequences of the chloroplast gene matKand the internal transcriber spacer (ITS) of ribosomal rDNA genes. DNA was extracted, and gene primers trnK3914 and trnK2R were used to amplify matK, and primers 1406F and ITS2 were used to amplify the ITS1 region between 18S and 5.8S ribosomal DNA units. Both matKand ITS were sequenced for each plant specimen and sequences were aligned to identify nucleotide diversity and detect SNPs. Variation in nucleotide sequence for either gene yielded similar dendrograms. Nucleotide variation among the Astilbeutilized in this study has allowed the development of SNP markers that may be useful for fingerprinting unknown hybrids or cultivars in the industry, and may be used for species alignment within the genus.

Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 300:91-103 (2005) - doi:10.3354/meps300091 Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina S. Derycke1,4,*, T. Remerie1,4, A. Vierstraete2,4, T. Backeljau3, J. Vanfleteren2,4, M. Vincx1, T. Moens1 1Department of Biology, Marine Biology Section, Ghent University, Krijgslaan 281, S8, 9000 Ghent, Belgium2Department of Biology, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium3Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium4CeMoFE, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium *Present address: Krijgslaan 281 Email: s.derycke@ugent.be ABSTRACT: An inverse correlation between dispersal ability and genetic differentiation among populations of a species is frequently observed in the marine environment. We investigated the population genetic structure of the free-living marine nematode Pellioditis marina. A total of 426 bp of the mitochondrial cytochrome oxidase subunit 1 (COI) gene were surveyed on a geographical scale of approximately 100 km during spring 2003. Nematodes were collected from 2 coastal locations in Belgium, and from 2 estuaries and a saltwater lake (Lake Grevelingen) in The Netherlands. Molecular variation was assessed with the single-strand conformation polymorphism (SSCP) method. In total, 32 different haplotypes were observed, and sequence divergence among 452 individuals ranged from 0.2 to 10.6%. We discovered 4 distinct mitochondrial lineages, with low divergences within the lineages (0.2 to 1.6%) and high divergences between the lineages (5.1 to 10.6%). The nuclear ribosomal ITS (internal transcribed spacer) region showed concordant phylogenetic patterns, suggesting that nematode species diversity may be considerably underestimated. Analysis of molecular variance (AMOVA) indicated a strong genetic differentiation among populations. The Lake Grevelingen population was clearly differentiated from all other populations, but genetic structuring was also significant within the Westerschelde and was correlated with gradients in salinity and pollution. The observed population genetic structure is in accordance with the limited active dispersal capacity of P. marina, but is at variance with its significant potential for passive dispersal. We therefore suggest that autecological characteristics, including short generation time, high colonization potential and local adaptation, may be at the basis of this nematode’s population genetic structure. KEY WORDS: Pellioditis marina · Single-strand conformation polymorphism · Cryptic species · Population genetic structure Full text in pdf format Supplementary Appendix PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 300. Online publication date: September 16, 2005 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2005 Inter-Research.

Molecular and Morphological Characterization of the Willow Rust Fungus, Melampsora epitea, from Arctic and Temperate Hosts in North America

Current taxonomy places all rust fungi that occur on willow (Salix spp.) in North America in one species complex, Melampsora epitea Thüm. Characteristics of M. epitea isolates from the Canadian arctic were compared to M. epitea isolates from temperate regions of North America. Sequences from internal transcribed spacer (ITS) regions of rDNA were obtained from urediniospores from rust-infected Salix leaves collected in the Canadian arctic and in Minnesota and compared. Phylogenetic analysis of nuclear ribosomal ITS regions indicated that arctic M. epitea samples were divergent from temperate M. epitea isolates, perhaps in part because all rusts examined diverged according to host species. Four urediniospore characteristics were examined: area, circularity (shape factor), major axis length and spine density. Statistically significant (P < 0.05) differences were observed for spine density among all host species except S. nigra and S. bebbiana. However major axis length differed between these species. These results represent the first evidence that arctic and temperate Melampsora species on Salix hosts in North America have evolved distinct molecular and morphological characters.