Well-supported Cluster Research Articles

Preponderant clonal evolution of Trypanosoma cruzi I from Argentinean Chaco revealed by Multilocus Sequence Typing (MLST)

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI.

Propionibacterium olivae sp. nov. and Propionibacterium damnosum sp. nov., isolated from spoiled packaged Spanish-style green olives.

Five strains of Gram-stain-positive bacteria were isolated from anomalous fermentations occurring in post-packaging of sealed airtight food-grade plastic pouches of Spanish-style green olives. These isolates could be grouped into two sets, which showed a similarity in their respective 16S rRNA gene sequences of 98.40 and 98.44 % with Propionibacterium acidipropionici NCFB 563 and 98.33 and 98.11 % with Propionibacterium microaerophilum M5(T), respectively, and a similarity of 99.41 % between them. The 16S rRNA gene phylogeny revealed that the isolates grouped into two statistically well-supported clusters separate from P. acidipropionici NCFB 563 and P. microaerophilum M5(T). Enzymic activity profiles as well as fermentation patterns differentiated these two novel bacteria from other members of the genus Propionibacterium. Finally, phenotypic, genotypic and phylogenetic data, supported the proposal of two novel species of the genus Propionibacterium, for which the names Propionibacterium olivae sp. nov. (type strain, IGBL1(T) = CECT 8061(T) = DSM 25436(T)) and Propionibacterium damnosum sp. nov. (type strain, IGBL13(T) = CECT 8062(T) = DSM 25450(T)) are proposed.

Enterococcus olivae sp. nov., isolated from Spanish-style green-olive fermentations.

Six strains of a hitherto unknown, Gram-stain-positive coccus were recovered from samples of Spanish-style green-olive fermentations. The 16S rRNA gene sequences from these isolates shared 98.7% and 98.5% of their nucleotide positions with those from Enterococcus saccharolyticus subsp. taiwanensis 812(T) and from E. saccharolyticus subsp. saccharolyticus ATCC 43076(T), respectively. The sequence of the rpoA gene in the isolates was 95% similar to that of E. saccharolyticus CECT 4309(T) ( = ATCC 43076(T)). The 16S rRNA and rpoA gene phylogenies revealed that the isolates grouped in a statistically well-supported cluster separate from E. saccharolyticus. Enzyme activity profiles as well as fermentation patterns differentiated the novel bacteria from other members of the Enterococcus genus. Finally, phenotypic, genotypic and phylogenetic data supported the identification of a novel species of the genus Enterococcus, for which the name Enterococcus olivae sp. nov. is proposed. The type strain is IGG16.11(T) ( = CECT 8063(T) = DSM 25431(T)).

First Report of Leaf Spot Caused by Alternaria alternata on Chinese Dwarf Banana in China.

The Chinese dwarf banana (Ensete lasiocarpum) is one of the ornamental bananas that belongs to Musaceae family. The plant is native to the southwestern China, where it grows semi-wild in the mountains between 1,500 and 2,500 m above sea level. During July 2011, a leaf spot disease on this plant was observed in the campus and parks in Kunming, Yunnan Province. The incidence level was about 22%, mainly on the old leaves. The leaf symptoms were irregular spots with gray to off-white centers surrounded by dark brown margins, and usually also surrounded by chlorotic halos. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected (95% ethanol for 3 min, 0.1% HgCl2 for 2 min, rinsed three times with sterile water), plated on potato sucrose agar (PSA), and incubated at 26°C under natural lights. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PSA that were black on the underside. The colonies were further identified as Alternaria sp. based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 5.26 to 30.26 μm long and 3.95 to 15.79 μm wide, averaging 10.21 (±3.17) × 20.02 (±5.75) μm (n = 50), with a beak length of 0 to 7.89 μm, and had 3 to 8 transverse and 0 to 3 longitudinal septa. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (1). The ITS region of isolate DY1 (GenBank Accession No. KF516556) was 572 bp in length. BLAST search revealed 99% identity with two Alternaria alternata isolates (JF440581.1 and GQ121322.2). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the isolate in a well-supported cluster with other A. alternata isolates. The pathogen was identified as A. alternate (Fr.:Fr.) Keissler based on the morphological characteristics and rDNA-ITS sequence analysis. To confirm pathogenicity, Koch's postulates were performed on detached leaves of E. lasiocarpum inoculated with mycelial plugs with ddH2O and agar plugs as a control. Leaf spots identical to those observed in the field developed in 9 days on the inoculated leaves but not on the control. The inoculation assay used three leaves, totaling 72 spots for control and 36 spots for inoculation. The experiments were repeated once. A. alternata was consistently re-isolated from the inoculated leaves. The symptom developed easier with wounds. To our knowledge, this is the first report of E. lasiocarpum leaf spot disease caused by A. alternata in China and the world.

Anatoecusspecies (Phthiraptera: Philopteridae) from Anseriformes in North America and taxonomic status ofAnatoecus dentatus and Anatoecus icterodes

AbstractAnatoecusCummings (Phthiraptera: Philopteridae) is a genus of chewing lice with four species infesting Anseriformes in North America:Anatoecus cygni(Denny),Anatoecus dentatus(Scopoli),Anatoecus icterodes(Nitzsch), andAnatoecus penicillatusKéler. Males ofA. dentatusandA. icterodesare distinguishable by their genitalia; however, there are no known anatomical characteristics to distinguish females.Anatoecus dentatusandA. icterodesare recorded from at least 55 of the same host species worldwide. The mitochondrial gene cytochrome c oxidase subunit I was examined from the fourAnatoecusspecies, specifically to examine the taxonomic status ofA. dentatusandA. icterodes. When sequences from these species were analysed using neighbour joining analysis,A. dentatusandA. icterodeswere recovered in a well-supported cluster. However,A. dentatusandA. icterodeswere mixed within the same cluster. The average interspecific genetic distance ofA. dentatusandA. icterodes(0.04%) was almost the same as the average intraspecific genetic distances ofA. dentatusandA. icterodes0.02% and 0.05%, respectively. Therefore, we formally synonymiseA. dentatusandA. icterodesasAnatoecus dentatus(new synonymy). In addition two new hosts forA. penicillatuswere recorded:Branta canadensis(Linnaeus) (Anseriformes: Anatidae) andChen caerulescens(Linnaeus) (Anseriformes: Anatidae).

First Report of Cylindrocarpon destructans var. destructans Causing Black Root Rot of Sanqi (Panax notoginseng) in China.

Sanqi (Panax notoginseng (Burk.) F. H. Chen) is planted on >10,000 ha in China and is a popular Chinese medicinal material (2). Black root rot is a recently identified but worsening problem on Sanqi since 2010 in Wenshan, China. Of the plant tubers examined from 185 ha, 8.5 to 27.4% were black with necrotic lesions. The base of leaves of infected plants had brown, sunken, necrotic lesions, and symptomatic plants had one to three chlorotic leaves. A fungus was isolated consistently from the basal leaves, bulb, and tubers of symptomatic plants. Six single-spore isolates were cultured on potato sucrose agar (PSA) at 25 ± 1°C in the dark. The mycelium of each culture was white initially on PSA, and then became rust-colored. The adaxial surfaces of the plates were black. Conidiophores were 13.6 to 167.3 × 1.4 to 21.8 μm (avg. 68.6 × 2.9 μm), single or with up to four levels of branching and two to three branches (or phialides) per level. The basal branches were often divergent, whereas the terminal branches were usually more appressed. Sporodochia were not present. Microconidia were 0-septate, 4.1 to 9.5 × 2.7 to 4.1 μm (avg. 8.2 × 2.9 μm). Conidia were 1- to 3-septate and occasionally 4-septate. One- to 3-septate conidia were clavate, with a truncate or slightly protruding conidial base, 9.2 to 40.8 × 3.5 to 6.8 μm (avg. 26.7 × 5.2 μm); whereas 4-septate conidia were 32.6 to 50.3 × 5.4 to 6.8 μm (avg. 40.9 × 6.5 μm). Chlamydospores were abundant, golden to brown, single or in chains or clumps, and up to 21.8 μm in diameter. PCR amplification was carried out for one isolate, RR926, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (4). Sequencing of the PCR product (GenBank Accession No. KC904953) revealed 99% similarity (99% coverage) with the ITS sequence of Cylindrocarpon destructans var. destructans (AM419065). Phylogenetic analysis (MEGA 4.1) using the neighbor-joining algorithm placed the isolate in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with AM419065. Therefore, the pathogen was identified as C. destructans (Zinssm.) Scholten var. destructans (teleomorph Ilyonectria radicicola (Gerlach & L. Nilsson) P. Chaverri & C. Salgado) based on morphological characteristics and rDNA-ITS sequence analysis (1,3). Pathogenicity tests of the six isolates were conducted on five 1-year-old and five 3-year-old plants/isolate. The roots of all plants were washed with sterilized water, and then surface-sterilized with 75% ethanol. Inoculum (1 ml of 106 conidia/ml) of each isolate was brushed onto the roots of each plant with a paintbrush. Inoculated plants were planted in pots in a mixture of sterilized quartz sand:vermiculite:pearlite (2:1:1, v/v). The pots were placed under black shadecloth. The roots of five 1-year-old and five 3-year-old plants were brushed similarly with sterilized water as control treatments. After 30 days, symptoms similar to those on the original diseased plants were observed on the roots of all plants inoculated with the six isolates. The roots of non-inoculated plants remained healthy. The experiment was repeated. The same pathogen was re-isolated from the inoculated plants, but no pathogen was isolated from roots of the control plants. C. destructans var. destructans is widely distributed in soils (1), but to our knowledge, this is the first report of this fungus causing black root rot of Sanqi in China.

Short-read DNA Sequencing Yields Microsatellite Markers for Rheum

Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.

A nuclear DNA based phylogeny of endemic sand dune ants of the genus Mycetophylax (Emery, 1913): How morphology is reflected in molecular data

Molecular methods havesubstantially advanced our knowledge about ant systematics in the past few years. Here, we infer the molecular phylogeny of sand dune ants of the genus Mycetophylax, Emery 1913 (Formicidae: Myrmicinae: Attini) using 730 base pairs of DNA sequences of the two nuclear genes longwave rhodopsin and wingless. Our analyses indicate that Mycetophylax is monophyletic, as suggested by its morphological characters. M. morschi, previously considered a species of Cyphomyrmex due to a scrobe-like impressed area on the head, forms a well-supported cluster with the two other species of Mycetophylax, M. conformis and M. simplex. Our analysis yields the first comprehensive phylogeny of Mycetophylax based on molecular data and includes specimens from localities within a wide distributional range as well as all species belonging to the genus following the recent taxonomic revision.

Evidence for two phylogenetic clusters within hepatitis C virus (HCV) genotype 2 inferred from analysis of complete coding sequences of 15 HCV strains

The aim of this study was to gain further insight into the evolution and classification of hepatitis C virus (HCV) by assessing the subtype distribution of 273 genotype 2 strains isolated from French blood donors from 1990 to 2010 and by determining complete coding sequences in subtype 2 strains. These classified into 7 of the established subtypes and into 15 additional lineages not yet assigned to a known subtype. Phylogenetic tree construction showed two well-supported clusters. Cluster 1 included most subtype 2 strains while cluster 2 included subtype 2l and one unassigned subtype 2. Full genome sequencing was performed on 15 genotype 2 strains belonging to both clusters, that is, one subtype 2b, two subtype 2c, three subtype 2i, two subtype 2j, one subtype 2k, two subtype 2l, and four unassigned strains. Genomes included a 9042- to 9108-nucleic acid open reading frame coding for a polyprotein comprising 3014-3036 amino acids. Mean nucleotide distances between subtypes belonging to the first cluster was 20.2 ± 1.4% while the mean distance between the two clusters was 25.9 ± 0.3%. Analysis indicated that the bifurcation between subtype 2l and other subtype 2 strains occurred early in the evolutionary process. Subtype 2l retained a genomic feature characteristic of non-genotype 2, that is, absence of the 60-nucleotide insertion in the NS5A region. This finding suggests that appearance and fixation of this insertion occurred late in the evolutionary history of HCV type 2 and that its absence is an ancestral feature of HCV.

Evolutionary Dynamics of West Nile Virus in the United States, 1999–2011: Phylogeny, Selection Pressure and Evolutionary Time-Scale Analysis

West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10−4 substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.

Phenotypic and genotypic characteristics of Phormidium-like cyanobacteria inhabiting microbial mats are correlated with the trophic status of running waters

Polyphasic analysis was carried out of natural samples and strains isolated from Phormidium mats, collected from different Spanish rivers with distinct water quality. 16S rRNA gene analysis separated five main clusters in a phylogenetic tree corresponding to five main, previously observed morphotypes. Two isolated strains and three natural samples had a relatively homogeneous morphology, which could correspond to Phormidium autumnale, and their sequences grouped in a well-supported cluster (morphotype–genotype I). However, ultrastructural differences were found among representatives from this cluster, and three clades could be separated in a further phylogenetic analysis. The samples belonging to the first clade were characteristic of hypertrophic waters, while representatives of the second grew in mesotrophic conditions, and those of the third in eutrophic waters. These results suggested that our representatives of morphotype–genotype I belong to three different species. According to our results and other published data, complementary information was obtained on the basis of morphological, phylogenetic and ecological criteria. Morphotype–genotype II, corresponding to Phormidium terebriforme, is a potential indicator of polluted waters. Oscillatoria tenuis, corresponding to morphotype–genotype III, is characteristic of eutrophic conditions. Phormidium mats identified as Phormidium corium (morphotype–genotype IV) were found to be characteristic of waters with low nutrient levels. Finally, morphotype–genotype V, identified as Phormidium aerugineo-caeruleum was dominant in oligo- to mesotrophic conditions.

Phylogenetic diversity of Rhizobium strains nodulating diverse legume species growing in Ethiopia

The taxonomic diversity of thirty-seven Rhizobium strains, isolated from nodules of leguminous trees and herbs growing in Ethiopia, was studied using multilocus sequence analyses (MLSA) of six core and two symbiosis-related genes. Phylogenetic analysis based on the 16S rRNA gene grouped them into five clusters related to nine Rhizobium reference species (99–100% sequence similarity). In addition, two test strains occupied their own independent branches on the phylogenetic tree (AC86a2 along with R. tibeticum; 99.1% similarity and AC100b along with R. multihospitium; 99.5% similarity). One strain from Milletia ferruginea was closely related (>99%) to the genus Shinella, further corroborating earlier findings that nitrogen-fixing bacteria are distributed among phylogenetically unrelated taxa. Sequence analyses of five housekeeping genes also separated the strains into five well-supported clusters, three of which grouped with previously studied Ethiopian common bean rhizobia. Three of the five clusters could potentially be described into new species. Based on the nifH genes, most of the test strains from crop legumes were closely related to several strains of Ethiopian common bean rhizobia and other symbionts of bean plants (R. etli and R. gallicum sv. phaseoli). The grouping of the test strains based on the symbiosis-related genes was not in agreement with the housekeeping genes, signifying differences in their evolutionary history. Our earlier studies revealing a large diversity of Mesorhizobium and Ensifer microsymbionts isolated from Ethiopian legumes, together with the results from the present analysis of Rhizobium strains, suggest that this region might be a potential hotspot for rhizobial biodiversity.

Cytogenetic and Molecular Analyses Reveal a Divergence between Acromyrmex striatus (Roger, 1863) and Other Congeneric Species: Taxonomic Implications

The leafcutter ants, which consist of Acromyrmex and Atta genera, are restricted to the New World and they are considered the main herbivores in the neotropics. Cytogenetic studies of leafcutter ants are available for five species of Atta and 14 species of Acromyrmex, both including subspecies. These two ant genera have a constant karyotype with a diploid number of 22 and 38 chromosomes, respectively. The most distinct Acromyrmex species from Brazil is A. striatus, which is restricted to the southern states of Santa Catarina and Rio Grande do Sul. Several cytogenetic and phylogenetic studies have been conducted with ants, but the karyotypic characterization and phylogenetic position of this species relative to leafcutter ants remains unknown. In this study, we report a diploid number of 22 chromosomes for A. striatus. The phylogenetic relationship between A. striatus and other leafcutter ants was estimated based on the four nuclear genes. A. striatus shared the same chromosome number as Atta species and the majority of metacentric chromosomes. Nuclear data generated a phylogenetic tree with a well-supported cluster, where A. striatus formed a different clade from other Acromyrmex spp. This combination of cytogenetic and molecular approaches provided interesting insights into the phylogenetic position of A. striatus among the leafcutter ants and the tribe Attini.

First Report of Leaf Spot Disease Caused by Colletotrichum gloeosporioides on Chinese Bean Tree in China.

Chinese bean tree, Catalpa fargesii f. duciouxii (Dode) Gilmour, is an ornamental arbor plant. Its roots, leaves, and flowers have long been used for medicinal purposes in China. During July 2010, severe outbreaks of leaf spot disease on this plant occurred in Kunming, Yunnan Province. The disease incidence was greater than 90%. The symptoms on leaves began as dark brown lesions surrounded by chlorotic halos, and later became larger, round or irregular spots with gray to off-white centers surrounded by dark brown margins. Leaf tissues (3 × 3 mm), cut from the margins of lesions, were surface disinfected in 0.1% HgCl2 solution for 3 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PDA, and were slightly brown on the underside of the colony. The hyphae were achromatic, branching, septate, and 4.59 (±1.38) μm in diameter on average. Perithecia were brown to black, globose in shape, and 275.9 to 379.3 × 245.3 to 344.8 μm. Asci that formed after 3 to 4 weeks in culture were eight-spored, clavate to cylindrical. The ascospores were fusiform, slightly curved, unicellular and hyaline, and 13.05 to 24.03 × 10.68 to 16.02 μm. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (2). Sequencing of the PCR products of DQ1 (GenBank Accession No. JN165746) revealed 99% similarity (100% coverage) with Colletotrichum gloeosporioides isolates (GenBank Accession No. FJ456938.1, No. EU326190.1, No. DQ682572.1, and No. AY423474.1). Phylogenetic analyses (MEGA 4.1) using the neighbor-joining (NJ) algorithm placed the isolate in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with other C. gloeosporioides isolates. The pathogen was identified as C. gloeosporioides (Penz.) Penz. & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld & H. Schrenk) based on the morphological characteristics and rDNA-ITS sequence analysis (1). To confirm pathogenicity, Koch's postulates were performed on detached leaves of C. fargesii f. duciouxii, inoculated with a solution of 1.0 × 106 conidia per ml. Symptoms similar to the original ones started to appear after 10 days, while untreated leaves remained healthy. The inoculation assay used three leaves for untreated and six leaves for treated. The experiments were repeated once. C. gloeosporioides was consistently reisolated from the diseased tissue. C. gloeosporioides is distributed worldwide causing anthracnose on a wide variety of plants (3). To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf spots on C. fargesii f. duciouxii in China.

AFLP analyses do not provide support for recognizing Brachyglottis saxifragoides as a species distinct from B. lagopus (Asteraceae; Senecioneae)

Historically, two morphologically similar species of rosette-forming Brachyglottis have been recognized by Flora writers as present on Banks Peninsula (Canterbury, New Zealand): B. lagopus (widespread throughout New Zealand) and B. saxifragoides (endemic to the Port Hills). We compared amplified fragment length polymorphism (AFLP) profiles from Banks Peninsula plants with those of plants identified as B. bellidioides, B. haastii and B. lagopus mostly from the Canterbury Southern Alps. The B. lagopus and B. saxifragoides plants from Banks Peninsula form a cluster that is distinct from those formed by B. bellidioides, B. haastii and B. lagopus populations from elsewhere in New Zealand. Although all B. saxifragoides specimens included in our analyses formed a well-supported cluster within the Banks Peninsula cluster, the lack of consistent morphological differences between this species and B. lagopus and the strong geographical clustering of AFLP profiles (rather than a clustering along current taxonomic lines) suggest that only one species is present on Banks Peninsula.

Branched Halymenia species (Halymeniaceae, Rhodophyta) in the Indo-Pacific region, including descriptions of Halymenia hawaiiana sp. nov. and H. tondoana sp. nov.

Several species in the red algal genus Halymenia from the Indo-Pacific have been described with branched thalli, toothed margins, spinose proliferations on the blade, and a firm gelatinous texture. Previous works have synonymized many of these morphologically similar species with H. durvillei. Our increased taxon sampling and molecular data indicate that the taxonomy of the Indo-Pacific Halymenia species is in need of revision, and that several aspects of taxonomies proposed by previous authors now seem unlikely. Thus, the aim of the present work was to analyse species delimitation in branched Halymenia species. Molecular and morphological data for specimens from the coral triangle and peripheral Indo-Pacific localities (East African coast, Hawaii) were used to understand species delimitation for selected branched Halymenia spp. Phylogenetic analyses based on 29 rbcL gene sequences grouped the specimens in four well-supported clusters at the species level with high p-distances (2.7–5.3%). After the morphological analysis, five features were retained as diagnostic to identify the four species studied. Our analyses led to the recognition and description of two new species, H. hawaiiana from the Hawaiian Islands (previously erroneously called H. formosa) and H. tondoana from the Philippines. In addition, H. harveyana (currently treated as a subspecies of H. floresii in Australia) is reassessed and recognized at the species level. Specimens with seven orders of branching and a thick cortex (70–150 µm) formed a monophyletic group, including sequences from previous work, with mostly well-supported branches and with high p-distances at the species level. We propose to call this group the H. durvillei complex until further reassessment is completed. None of the sequences studied here grouped with H. floresii from the Mediterranean, suggesting that previous Indo-Pacific reports of the species were erroneous.

Evolutionary classification: A case study on the diverse plant genus Ranunculus L. (Ranunculaceae)

Evolutionary classification attempts to integrate information on shared ancestry, evolutionary process and phenetic information into the taxonomic concept. Here we exemplify this concept on the monophyletic, species-rich and cosmopolitan plant genus Ranunculus. Previous classifications have rendered almost all traditional sections as polyphyletic, and a modern revision based on phylogenetic principles was so far lacking. Maximum parsimony and Bayesian inference analysis of a combined nuclear (ITS of nrDNA) and plastid DNA dataset (matK/trnK, and psbJ–petA) provided a phylogenetic framework for the genus with nine well-supported subclades. Neighbor Net analysis revealed a reticulate data structure within two subclades with frequent polyploidy and/or hybridization. Character evolution was studied by McClade reconstructions of morphological data mapped on to the molecular tree topology. Morphological characters show a mosaic-like distribution, but express several shared states congruent to molecular clades. A total evidence approach (TE) based on the combined morphological and molecular dataset suggests a subdivision of Ranunculus into a paraphyletic, temperate to arctic group of five subclades (subg. Auricomus), and a temperate to subtropical clade with four subclades (subg. Ranunculus). Infrageneric classification of two subgenera and 17 sections is based on both monophyly s.l. as evident from TE and a minimum of shared morphological characters. Six subclades have shared morphological or karyological features (sects. Auricomus, Flammula, Oreophili, Polyanthemos, Ranunculus, Thora, and Trisecti). One subclade was subdivided into three smaller clades according to morphological data (sects. Epirotes, Leucoranunculus, Ranuncella, Aconitifolii). In the case of reticulate evolution and uncertain ancestry we accept well-supported genetic clusters with shared morphological features, as revealed by Neighbor Net analysis (sections Batrachium, Hecatonia, Pseudadonis). Character evolution connected to ecological shifts characterizes the paraphyletic section Ranunculastrum, the holophyletic section Euromontani (sect. nov.), and the monotypic sect. Echinella. The information content of our classification is compared to alternative concepts.

Genetic Structure of <i>Ascaris</i> Roundworm in Japan and Patterns of Its Geographical Variation

Ascaris roundworm isolates from Japan and central Europe were examined by sequencing analyses to better understand geographically induced nucleotide variation and genotype distribution according to host. Three well-supported clusters (denoted as A, B, C) were identified by generating cox1 sequences of mtDNA from these regions. Among 5 pig isolates collected in eastern Honshu, Japan, in 2010, 3 carried DNA characteristics for cluster A and 2 corresponded with the characteristics of cluster B. The sequence of the human isolate JH1 from north-central Honshu, fixed in formalin since 1972, conformed to the characteristics of cluster A. Differential analysis of ribosomal ITS1 region revealed the JH1 isolate sequence profile of Ascaris lumbricoides. Cluster C, which was the most distinguish cluster, was formed by reference Slovak isolates and has been so far found almost exclusively in European pigs. A fluctuating prevailing distribution of A and B lineages in human and pig hosts in different territories of the world and the global distribution of several haplotypes indicate their establishment before secondary differentiation in a given region due to host affiliation. The protocol established for DNA isolation from formalin-fixed specimens using the modified procedure with the Qiagen extraction set can be used as a tool for retrospective studies in ascarid helminths when only archival specimens are available.

Multilocus sequence analysis provides basis for fast and reliable identification of Vibrio harveyi-related species and reveals previous misidentification of important marine pathogens

Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. Phenotypic tests and 16S rRNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. This study used multilocus sequence analysis to identify 36 V. harveyi-like isolates obtained from a wide range of sources in Australia and to re-evaluate the identity of important pathogens. Phylogenies inferred from the 16S rRNA gene and five concatenated protein-coding genes ( rpoA– pyrH– topA– ftsZ– mreB) revealed four well-supported clusters identified as V. harveyi, V. campbellii, V. rotiferianus and V. owensii. Results revealed that important V. campbellii and V. owensii prawn pathogens were previously misidentified as V. harveyi and also that the recently described V. communis sp. nov. is likely a junior synonym of V. owensii. Although the MLSA topologies corroborated the 16S rRNA gene phylogeny, the latter was less informative than each of the protein-coding genes taken singularly or the concatenated dataset. A two-locus phylogeny based on topA- mreB concatenated sequences was consistent with the five-locus MLSA phylogeny. Global Bayesian phylogenies inferred from topA- mreB suggested that this gene combination provides a practical yet still accurate approach for routine identification of V. harveyi-related species.

Molecular and chemical characterization of the most widespread Ocimum species

DNA fingerprinting (AFLP) and chemical analyses of essential oils were utilized to define the extent of variation existing in the genus Ocimum. Research was carried out on 22 Ocimum accessions representing seven species. Concerning the essential oil composition of all investigated accessions, 115 compounds were identified. UPGMA cluster analysis, based on Euclidian distances of essential oil constituents between all pairs of accessions, showed four well-supported clusters (O. tenuiflorum, O. basilicum/O. africanum, O. basilicum, and O. americanum/O. africanum). Relating to the essential oil composition of all of the investigated accessions, 17 compounds were identified as the main ones, and according to them 13 chemotypes were determined. AFLP relationships were determined by neighbor-joining (NJ) cluster analysis based on Dice’s distance matrix and by maximum parsimony (MP) analysis. O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum represented four clusters supported with high bootstrap values. A neighbor-net diagram allowed the visualization of apparently conflicting data by revealing relationships between genotypes and chemotypes. Concerning the O. africanum species, two distinct chemotypes, geranial/neral (accession 11) and estragol (accession 10), have been established, while all the studied O. americanum accessions belong to the geranial/neral chemotype. This could be additional evidence that O. americanum is one of the parents of O. africanum. Furthermore, the fact that the O. africanum accession (10) as well as O. basilicum ‘Purpurascens’ and O. basilicum ‘Erevanskii’ accessions belong to the estragol chemotype supports the theory that O. africanum is one of the parents of these two O. basilicum accessions.