Ribosomal DNA ITS Research Articles

Deladenus prorsus n. sp. (Nematoda: Neotylenchidae) in dunnage wood from Malaysia intercepted in Ningbo, China

Deladenus prorsus n. sp., isolated from dunnage wood originating from Malaysia and intercepted in Ningbo port, P.R. China, is described and illustrated. The dunnage wood material appeared to have holes resembling the typical exit holes of wood-boring insects and have associated blue stain. The new species is characterised by the presence of both the mycophagous and infective forms (Deladenus consists of mycetophagous-only forms and those with known female dimorphism), the body length of 814 (670-1147) and 898 (812-979) μm for mycetophagous males and females, respectively, and 1129 (1114-1352) μm for the infective female, and by the very anterior position of the excretory pore which is situated at 30.7 (24.4-33.0), 30.6 (25.4-35.6) and 37.9 (27.3-39.7) μm for the mycetophagous males and females and infective females, respectively, and the distance that the excretory pore is located anterior to the hemizonid which is 82 (77-90), 74 (54-92) and 86 (64-106) μm for the mycetophagous males and females and infective females, respectively. The diagnostic value of the excretory pore and the hemizonid of the species of the genus are discussed. The ribosomal DNA ITS region was sequenced and analysed.

Application of PCR in Serum Samples for Diagnosis of Paracoccidioidomycosis in the Southern Bahia-Brazil

Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail.

Notes on the GenusAporpium(Auriculariales, Basidiomycota), with a New Species from Temperate Europe

A new polypore, Aporpium macroporum Niemelä, Spirin & Miettinen, is described on the basis of material from Finland, Poland (Białowieza National Park; type locality), European Russia, Belarus, Estonia, and Latvia. It grows primarily on fallen aspen trees and prefers old forests with abundant coarse woody debris. Aporpium caryae is an American taxon, and its European kin is A. canescens; A. macroporum differs from them in having wider pores, softer consistency, and paler colours. The spores of the new species are wider than in A. canescens, and longer than in A. caryae. The three species can be distinguished by their ribosomal DNA ITS sequences. We briefly discuss the heterobasidioid genera Aporpium, Elmerina and Protomerulius.

Morphological and molecular differentiation of smooth-hound sharks (GenusMustelus, Family Triakidae) from the Gulf of California

Summary The genus Mustelus is the most species-rich of the widespread family Triakidae whereby its taxonomy and systematics have been historically challenging. They represent a significant fraction of the shark catches of small-scale fisheries in the Gulf of California. In order to provide information useful for their management and conservation, the morphological and genetic distinction of the four species found in the northern Gulf of California (M. albipinnis, M. californicus, M. henlei and M. lunulatus) were analyzed. Discriminant analysis of 10 morphometric variables placed each species in a distinct and significantly different region of multivariate morpho-space. The variables contributing most to their distinction were inter-nostril width, mouth length, upper and lower labial furrow length, and mouth width. Restriction fragment length polymorphisms (PCR-RFLP) of the nuclear ITS2 ribosomal DNA (rDNA) confirmed that each species represents a genetically cohesive and independent evolutionary lineage. In spite of the difficulty in differentiating these Mustelus species, a few cephalic measurements are useful to separate them. A PCR-RFLP assay (using RsaI and MspI on ITS2 rDNA amplicons) is also proposed for the molecular differentiation of these commercially harvested smooth-hound sharks, constituting the first molecular marker available for their identification. These data provide morphological and genetic tools that can be used to improve their management and conservation.

한국산 피막이속(Hydrocotyle L.) 식물의 분자계통학적 연구

Phylogenetic analyses were conducted to evaluate relationships of 5 taxa of Korean Hydrocotyle, H. spp. found in the Ulleung island including one outgroup (Centella asiatica). The molecular phylogenetic methods based on nuclear ribosomal DNA ITS region and cpDNA trnH-psbA region. Centella asiatica was used outgroup for analysis. As the result, genus Korean Hydrocotyle were grouped by 94% bootstrap value. Korean Hydrocotyle was grouped by four clades; Clade Ⅰ-H. maritima, H. sibthorpides and H. yabei clade Clade Ⅱ- H. nepalensis clade clade Ⅲ- H. ramiflora clade clade Ⅳ- H. spp. clade. H. maritima, H. sibthorpides and H. yabei was not distinguished, seperately. H. spp. was distinctly distinguished other Korean Hydrocotyle.

Molecular characterisation of Galba truncatula, Lymnaea neotropica and L. schirazensis from Cajamarca, Peru and their potential role in transmission of human and animal fascioliasis

BackgroundHuman and animal fascioliasis is emerging in many world regions, among which Andean countries constitute the largest regional hot spot and Peru the country presenting more human endemic areas. A survey was undertaken on the lymnaeid snails inhabiting the hyperendemic area of Cajamarca, where human prevalences are the highest known among the areas presenting a "valley transmission pattern", to establish which species are present, genetically characterise their populations by comparison with other human endemic areas, and discuss which ones have transmission capacity and their potential implications with human and animal infection.MethodsTherefore, ribosomal DNA ITS-2 and ITS-1, and mitochondrial DNA 16S and cox1 were sequenced by the dideoxy chain-termination method.ResultsResults indicate the presence of three, morphologically similar, small lymnaeid species belonging to the Galba/Fossaria group: Galba truncatula, Lymnaea neotropica and L. schirazensis. Only one combined haplotype for each species was found. The ITS-1, 16S and cox1 haplotypes of G. truncatula are new. No new haplotypes were found in the other two species. This scenario changes previous knowledge, in which only L. viator (= L. viatrix) was mentioned. Galba truncatula appears to be the most abundant, with high population densities and evident anthropophyly including usual presence in human neighbourhood. Infection by Fasciola hepatica larval stages were molecularly confirmed in two populations of this species. The nearness between G. truncatula populations presenting liver fluke infection and both human settings and schools for children, together with the absence of populations of other lymnaeid species in the locality, suggest a direct relationship with human infection.ConclusionsThe geographical overlap of three lymnaeid species poses problems for epidemiological studies and control action. First, a problem in classifying lymnaeid specimens in both field and laboratory activities, given their transmission capacity differences: G. truncatula mainly involved in transmission to humans, L neotropica typically responsible for livestock infection, and L. schirazensis unable for transmission. Although several phenotypic characteristics may be helpful for a preliminary specimen classification, a definitive classification can only be obtained by marker sequencing. Aditionally, L. schirazensis increases the confusion, owing to its ability to mix with other Galba/Fossaria species and distort fascioliasis data such as transmission capacity and infection susceptibility. Second, a problem for epidemiological analysis, surveillance and control by methods as mathematical modelling and Remote Sensing - Geographical Information Systems. In Cajamarca, low resolution mapping may be insufficient, as already verified in Andean areas where different lymnaeid species overlap.

Phylogenetic analysis of rDNA sequences indicates that the sequestrate Amogaster viridiglebus is derived from within the agaricoid genus Lepiota (Agaricaceae)

The rare sequestrate fungus Amogaster viridiglebus is known from only one collection in California where it was discovered among Populus roots. Based on sporocarp coloration and spore morphology, this sequestrate taxon was putatively considered to be an ectomycorrhizal member of the Boletales. However, no molecular data were previously available to definitively determine the closest relatives of this fungus. Here we revisit the morphology of Amogaster viridiglebus and present a phylogenetic analysis based on ITS and 28S ribosomal DNA. Our phylogeny indicates that Amogaster viridiglebus is nested in the genus Lepiota, suggesting that this rare species has a saprobic trophic mode and does not form ectomycorrhizae with plants. A new combination, L. viridigleba, is made based on these phylogenetic results.

Barcode Identifiers as a Practical Tool for Reliable Species Assignment of Medically Important Black Yeast Species

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Starting from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives.

Population structure, mitochondrial polyphyly and the repeated loss of human biting ability in anopheline mosquitoes from the southwest Pacific.

Australia and New Guinea contain high levels of endemism and biodiversity, yet there have been few evaluations of population-level genetic diversity in fauna occurring throughout the Australo-Papuan region. Using extensive geographical sampling, we examined and compared the phylogenetic relationships, phylogeography and population structure of Anopheles farauti, An. hinesorum and An. irenicus throughout their ranges in the southwest Pacific using mitochondrial (mtDNA COI) and nuclear (ribosomal protein S9 and ribosomal DNA ITS2) loci. Phylogenetic analyses suggest that the ability to utilize humans as hosts has been lost repeatedly, coincident with independent colonizations of the Solomon Islands. As some of the species under investigation transmit malaria in the region, this is a medically important finding. Maximum likelihood and Bayesian phylogenetic analyses of nuclear loci also showed that the three species are monophyletic. However, putative introgression of An. hinesorum mtDNA onto a nuclear background of An. farauti was evident in populations from Queensland, Torres Strait and southern New Guinea. Haplotype networks and pairwise FST values show that there is significant genetic structure within New Guinea and Australia in both An. farauti and An. hinesorum, consistent with a long-term history of low gene flow among populations.

First record of Ceratocystis fimbriata on Carapa guianensis

Crabwood or andiroba (Carapa guianensis) is a medium to large tree belonging to the family Meliaceae, widespread in tropical South America, used for timber, furniture, and oil from seeds for cosmetic and ethnomedicinal purposes. Despite its economic importance only one disease caused by Pestalotiopsis macrochaeta has been reported (Halfeld-Vieira & Nechet, 2006). In August 2008, during the course of surveys in a native forest in São João da Baliza municipality in Roraima state, Brazil, dying seedlings of C. guianensis were observed with symptoms of a fungal infection on stems (Fig. 1) and petioles. Only one fungus species was isolated from perithecia present in infected tissue, forming olive brown cultures on potato dextrose agar (PDA). The following morphological features were observed. Perithecia were dark brown, globose, 112-200 μm, neck erect, 431-680 μm, with divergent ostiolar hyphae (Fig. 2A, 2B); perithecial width at the base was 27-34 μm and 20 μm at the apex. Acospores were “hat” shaped, hyaline, 5-7 times 4-5 μm (Fig. 2C). The anamorph corresponded to Chalara with hyaline, cylindrical, catenulate endoconidia with truncate ends, 10-20 times 3.7-5 μm; chlamydospores were pale to dark brown, ovoid, thick-walled, 10-15 times 10-12 μm (Fig. 2D). Based on these morphological characteristics the fungus was identified as Ceratocystis fimbriata (Wingfield et al., 1993). To confirm the identity of the pathogen, the internal transcribed spacer (ITS) region of a representative isolate was amplified using ITS 1 and ITS 4 universal primers and sequenced (GenBank Accession No. JN051277). Ribosomal DNA-ITS sequence data were found to have up to 98% identity with C. fimbriata. To fulfil Koch's postulates, pathogenicity tests were performed in a greenhouse on two-month-old C. guianensis seedlings. Stems were wounded with one superficial puncture with a needle tip, a mycelium plug inserted and the wound covered with Parafilm. A PDA disk was used as control and each treatment consisted of six plants kept under greenhouse conditions. Seven days later the Parafilm was removed and the progress of the symptoms was evaluated. After 10 days, the pathogen was re-isolated from the stem lesions only on plants inoculated with mycelium plugs, presenting symptoms and signs similar to those occurring in the original infection. The culture of C. fimbriata was deposited in the fungal collection of Embrapa Roraima, a unit of the Brazilian Agricultural Research Corporation (access number CPAFRR515). Ceratocystis fimbriata is a native soilborne pathogen in some parts of Brazil and causes mortality in many economically important plants. In recent years, significant damage on Eucalyptus and Tectona grandis (teak) by this pathogen has been reported (Ferreira et al., 2010; Firmino et al., 2012). These exotic species are commonly planted in agroforestry systems with C. guianensis in Brazil. The high number of seedlings of C. guianensis affected by this pathogen observed in the field indicates its potential to have a significant impact on natural populations of this host and to be a threat to this species in agroforestry systems.

Identification of Spoilage Fungus from Strawberry Fruit by PCR

Strawberry fruit easily suffers from fruit disease caused by fungus infection because of its great susceptibility to microbial spoilage. In present paper the fungus pathogen was isolated from infected strawberry fruits. The molecular method was used to identify the species of the fungus. The internal transcribed spacer (ITS) region was amplified by PCR with the pair of ITS universal primers after extracting the genomic DNA from the isolate. The sequence alignment results indicated that the identity was 95% between the strain F1 and the fungus(GenBank: FN667943). Then the phylogenetic tree was constructed based on the sequence of ITS region of the fungal rRNA operon. According to the sequences of ribosomal DNA-ITS and phylogenetic analysis, the fungi was preliminaryly identified as Trichocomaceae. So the pathogen isolated from the infected strawberry fruits in Tianyi strawberry ecology located in Changping district, Beijing, China is a species of Trichocomaceae.

Phylogenetic placement of the Dominican Republic endemic genusSarcopilea(Urticaceae)

AbstractSince the first collection in 1910, botanists have recognizedSarcopilea(Urticaceae) as a dramatic example of convergence with succulent woody members of Crassulaceae. Phylogenetic analyses based on nuclear ribosomal DNA ITS and thetrnL–Fnon–coding plastid DNA regions placeSarcopileain a Neotropical clade ofPilea. A study of the cystolith, pollen and achene morphology, and of hydathode, hydrenchyma, and stomata position was also performed, confirming the molecular results. Across Rosales, leaf succulence is restricted to the generaPileaandSarcopilea. Furthermore, all succulent species ofPileahave an abaxial water–storage hydrenchyma layer in their leaves, a condition unique to the genus. The conditions of adaxial stomata (epistomatic, hyperstomatic), as well as hydathodes restricted to the submarginal abaxial leaf surface, were found in all Neotropical leaf–succulent species. Carbon isotope–ratio mass spectroscopy showsSarcopileato have C3photosynthesis, therefore Crassulacean acid metabolism is still unknown in Rosales. The impact of molecular systematics of Caribbean endemic genera is discussed. In light of the new phylogenetic placement ofSarcopilea, a replacement name is provided (Pilea fairchildiana).

DNA bacording of Verbenaceae medicinal plant by using ITS2 and psbA-trnH region

To evaluate the ability of several hotspot candidate sequence of DNA barcodes for identifying medicinal plant species in Verbenaceae. Using universal primers, three chloroplast sequences, psbA-trnH, rbcL, matK, two nuclear ribosomal DNA ITS2 and ITS were amplified and sequenced. PCR amplification and sequencing efficiency, intra- and inter-specific variation, barcoding gap and identification efficiency (with BLAST 1 and Nearest Distance methods) were used to evaluate these loci. The rate of successful amplification using matK was too low to further analyze, and the rate of successful amplification and sequencing using psbA-trnH, ITS, and ITS2 and rbcL sequence was 83.6%, 83.6%, 96.4%, 98.2%, respectively. The rate of successful identification using psbA-trnH, ITS, and ITS2 was 100% at the species level except that rbcL was 77.8%, 75.9% for 55 samples belonging to 32 species, but ITS2 did better in intra- and inter-specific variation, barcoding gap than the other loci. The rate of successful identification of ITS2 was 89.5%, 87.6% even when joining the date of 165 samples from GenBank. It proposes that the combination of ITS2 and psbA-trnH senquence is promising for the identification of the species in Verbenaceae.

Antimicrobial activity of endophytic fungi isolated from Dendrobium species in Southwestern China

To isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities. Fungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA. Ninety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum. Endophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.

Molecular and morphological identification of larval opecoelids (Digenea: Opecoelidae) parasitising prosobranch snails in a Western Mediterranean lagoon

In a study of the digeneans parasitising molluscs in the Els Alfacs lagoon (Ebro Delta, Western Mediterranean) we found heavy infections with sporocysts emitting two types of cotylocercous cercariae in the prosobranch trochid gastropod Gibbula adansonii and with metacercariae in the prosobranch nassariid gastropod Cyclope neritea. A comparative analysis using ITS ribosomal DNA sequences from these larval stages and published sequences of 17 larval and adult opecoelid stages allowed us to elucidate the life-cycle of Macvicaria obovata and to confirm the identification of Cainocreadium labracis based on cercarial morphology. We provide molecular evidence for the identification and the first detailed morphological descriptions of the intramolluscan larval stages of the two opecoelid species as well as partial 28S rDNA sequences to aid future studies on systematic relationships within the Opecoelidae.

Molecular Demonstration ofTrypanosoma evansiandTrypanosoma lewisiDNA in Wild Rodents from Cambodia, Lao PDR and Thailand

In this study, we investigated the molecular evidence of Trypanosoma evansi in wild rodents from Cambodia, Lao PDR and Thailand. Between November 2007 and June 2009, 1664 rodents were trapped at eight sites representative of various ecological habitats. Of those animals, 94 were tested by direct microscopic blood examination, 633 using the Card Agglutination Test for Trypanosomes (CATT/T.evansi) and 145 by Polymerase Chain Reaction (PCR) with two sets of primers: TRYP1 (amplifying ITS1 of ribosomal DNA of all trypanosomes) and TBR (amplifying satellite genomic DNA of Trypanozoon parasites). Using TRYP1, based on the size of the PCR products, 15 samples from the three countries were positive for Trypanosoma lewisi (two were confirmed by sequencing), and three were positive for Trypanozoon (one was confirmed by sequencing and three by TBR primers); the specificity of the primers failed as rodent DNA was amplified in some cases. Using TBR, six samples were positive for Trypanozoon (one was confirmed by sequencing); as T.evansi is the only species of the Trypanozoon sub-genus possibly present in Asian rodents, these results confirmed its presence in rodents from Thailand (Rattus tanezumi) and Cambodia (R.tanezumi, Niviventer fulvescens & Maxomys surifer). Further investigations are necessary to establish the situation in Lao PDR. None of the 16 samples most strongly positive to the CATT proved to be positive for Trypanozoon by PCR. The merits of the CATT for such studies were not confirmed. Studying the urban and rural circulation of these parasites in rodents will enable an evaluation of human exposure and infection risk, as human infections by T.evansi were recently described in India and by T.lewisi in India and Thailand. As sequencing PCR products is expensive, the development of new molecular and serological tools for rodents would be very useful.

First Report of Leaf Blight of Taro (Colocasia esculenta) Caused by Phytophthora colocasiae in Ghana.

Taro (Colocasia esculenta (L.) Schott) is an important food crop cultivated for its edible tubers in Ghana. In May 2009, outbreaks of a destructive leaf disease were observed on several taro farms in the Atiwa, East-Akim, Fanteakwa, West-Akim, and New Juaben districts of the Eastern Region of Ghana. Symptoms began on leaves as small, brown, water-soaked lesions that enlarged and coalesced into large lesions with yellow exudate, ultimately leading to the defoliation and death of plants. Symptoms were suggestive of taro leaf blight (TLB) caused by Phytophthora colocasiae Raciborski (3). By August 2010, the disease had spread to other taro-growing regions in Ghana. To identify the pathogen, leaf tissue from lesion margins were excised and plated on carrot agar and V8 selective media for Phytophthora and incubated at 27°C for 5 days (2). Growth from diseased tissue was used for morphological characterization. Sporangia were ovoid, hyaline, papillate, caducous, 30 to 60 × 17 to 28 μm, and pedicels were 3.5 to 10 μm long. Genomic DNA was isolated from pure cultures of two isolates, PCg11-2 from Oseim (6°15'N, 0°27'E) and PCg11-5 from Oyoko (6°8'N, 0°17'W). Ribosomal DNA ITS1, 5.8S and ITS2 were amplified by PCR using the ITS1 and ITS4 primers (4). The resultant 784-bp amplicons were sequenced (GenBank Accession Nos. JN662439 and JN662440). Sequences of both isolates were identical. A BLASTn search of these sequences revealed maximum homology of 99% with the sequence of P. colocasiae strains from blighted taro leaves in Nigeria (GenBank Accession No. HQ602756), Hawaii (GenBank Accession No. GU258997), and several strains in Asia and the South Pacific. On the basis of morphological characteristics and nucleotide homology, the isolates were identified as P. colocasiae. To fulfill Koch's postulates, 30 leaf discs from 3-month-old plants were inoculated with 10 μl of a suspension of 3 × 105 zoospores per ml (2). Leaf discs were incubated in the dark at 27°C on wet foam in plastic trays for 5 days. All inoculated discs developed blight symptoms similar in appearance to those observed on diseased taro in the fields. Control discs remained asymptomatic. P. colocasiae was reisolated from leaf discs and its identity confirmed by morphological characteristics. To our knowledge, this is the first report of TLB and P. colocasiae in Ghana. Occurrence of TLB was recently reported in Nigeria (1). The recent occurrence of TLB in both Nigeria and Ghana threaten the taro-growing regions of West and Central Africa. Disease surveys and a management strategy that includes resistant varieties are urgently needed.

The phylogeny of selected Phylloporus species, inferred from NUC-LSU and ITS sequences, and descriptions of new species from the Old World

The phylogeny of Phylloporus (Boletaceae) has not been well studied, and the taxonomic relationships of this genus have varied considerably among authors. The following study presents phylogenetic relationships of Phylloporus based on two nuclear ribosomal DNA regions, ITS and LSU. The ITS dataset includes 39 collections and the LSU dataset contains 50 collections of Phylloporus. A combined analysis of both genes did not resolve the deeper nodes in the phylogeny, but the results suggest that Phylloporus is monophyletic and a sister group of the Xerocomus subtomentosus group. The lamellate hymenophore configuration is a synapomorphy that distinguishes Phylloporus from the other genera in the family. The placement of a lamellate genus within Boletaceae suggests that hymenophore evolution is not well understood in the family. This is the first phylogeny of Phylloporus and includes 20 species from different geographic regions. Six taxa of Phylloporus from the Old World are here presented. Phylloporus cyanescens is a new combination for an Australasian taxon formerly named as a variety of P. bellus (Massee) Corner. Phylloporus pumilus is described from Indonesia, and 4 species are described from Thailand: P. castanopsidis, P. dimorphus, P. infuscatus, and P. rubiginosus.

Enterocytozoon bieneusiIdentification Using Real-Time Polymerase Chain Reaction and Restriction Fragment Length Polymorphism in HIV-Infected Humans from Kinshasa Province of the Democratic Republic of Congo

Objective. To determine the prevalence and the genotypes of Enterocytozoon bieneusi in stool specimens from HIV patients. Methods. This cross-sectional study was carried out in Kinshasa hospitals between 2009 and 2012. Detection of microsporidia including E. bieneusi and E. intestinalis was performed in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI) in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM) was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n = 19) among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3%) and 5 type IV strains of E. bieneusi (26.3%) were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo), and the concurrence of type I and type IV strains.

Taxonomical notes on Stachys sect. Eriostomum (Lamiaceae) in Turkey

As a part of a revisional study of Stachys L. sect. Eriostomum (Hoffmanns. & Link) Dumort. (Lamiaceae), 4 taxa [S. tymphaea Hausskn., S. thracica Davidov, S. cretica L. subsp. cretica, and S. cretica L. subsp. salviifolia (Ten.) Rech.f.] were recorded for the first time from Turkey. S. cretica subsp. trapezuntica Rech.f., which was described based on 3 syntypes [Bourgeau 695; Sintenis 3432 (designated here as the lectotype); Handel-Mazzetti 324] near Trabzon, had not been collected again since 1907. It was found in 2 syntype localities from Trabzon in 2010 during this study, for the first time in 103 years. For 2 taxa, the categories of other authors were accepted, as opposed to those in Flora of Turkey: S. germanica L. subsp. bithynica (Boiss.) R.Bhattacharjee (a subspecies) was replaced with S. bithynica Boiss. as a species, and S. balansae Boiss. & Kotschy subsp. carduchorum R.Bhattacharjee (a subspecies) was replaced with S. carduchorum (R.Bhattacharjee) Rech.f. as a species. In one taxon, the category of Haussknecht was accepted: S. tymphaea Hausskn. The status of S. libanotica Benth. var. minor Boiss. was changed to S. minor (Boiss.) Akçiçek & Dirmenci comb. et stat. nov. One species (S. ehrenbergii Boiss.) was removed from the flora of Turkey because it was mistakenly reported to grow in Turkey. Descriptions, diagnostic characters, detailed illustrations, distribution maps, and taxonomic comments for these taxa are presented. A phylogenetic analysis using the ITS of the nuclear ribosomal DNA of the examined taxa further confirmed the rearrangements suggested.