Atomic force microscopy investigations of heterogeneities in the adhesion energies measured between pathogenic and non-pathogenic Listeria species and silicon nitride as they correlate to virulence and adherence

Atomic force microscopy (AFM) was used to probe heterogeneities in adhesion energies measured between pathogenic and non-pathogenic species of Listeria and silicon nitride in water at four levels. Adhesion energies were quantified on individual bacterial cells (cell level), bacterial cells that belonged to an individual Listeria strain but varied in their cultures (strain level), bacterial cells that belonged to an individual Listeria species but varied in their strain type (species level) and on bacterial cells that belonged to the Listeria genus but varied in their species type (genus level). To quantify heterogeneities in the adhesion energies, a heterogeneity index (HI) was defined based on quantified standard errors of mean. At the cell level, spatial variations in the adhesion energies were not observed. For the strain, species, and genus levels, the HI increased with increased adhesion energies. At the species level, the HI increased with strain virulence.     

Genetic Relationships Among 22 Taxa of Bamboo Revealed by ISSR and EST-Based Random Primers

Genetic relationships among 22 taxa of bamboo were evaluated using 12 inter simple sequence repeats (ISSR) and four expressed sequence tag (EST)-based random primers, resulting in amplification of 220 loci. The grouping of species based on Jaccard's similarity matrix using UPGMA and principal coordinate analysis agreed with earlier published reports on molecular phylogenetic studies in bamboos with few deviations. In the dendrogram, however, species of one genus were placed in different clusters along with members of other genera. This calls for correct taxonomic delineation at the genus and species level using both vegetative and reproductive characters and correlation of molecular data with morphologically definable taxonomic groupings at the proper taxonomic level.     

裸盖菇属的真菌鉴定及分子系统学初探

裸盖菇属(Psilocybe)的许多真菌含有神经致幻型毒素,这些毒素被中国卫生部列为A类管制药品。在药检时,这些真菌样品通常是粉末。因此,仅依靠形态分类鉴定该类真菌非常困难。研究采用ITS序列分析的方法鉴定该类真菌并初步探讨了该属种间的系统发育关系。由系统发育树推断Psilocybe属可能是多源进化的。通过序列分析可以鉴定真菌样品为Psilocybe属。     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

First molecular phylogeny of the pantropical genus Dalbergia: implications for infrageneric circumscription and biogeography

The genus Dalbergia with c. 250 species has a pantropical distribution. In spite of the high economic and ecological value of the genus, it has not yet been the focus of a species level phylogenetic study. We utilized ITS nuclear sequence data and included 64 Dalbergia species representative of its entire geographic range to provide a first phylogenetic framework of the genus to evaluate previous infrageneric classifications based on morphological data. The phylogenetic analyses performed suggest that Dalbergia is monophyletic and that it probably originated in the New World. Several clades corresponding to sections of these previous classifications are revealed. Taking into account that there is not a complete correlation between geography and phylogeny, and the estimation that the Dalbergia stem and crown clades are 40.4–43.3 mya and 3.8–12.7 mya, respectively, it is plausible that several long distance dispersal events underlie the pantropical distribution of the genus.     

Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.     

Phylogeny of the European species of the genus Pellia (Hepaticae; Metzgeriales) based on the molecular data from nuclear tRNA(Leu)(CAA) intergenic sequences

Species of the genus Pellia are similar to such an extent that their proper recognition based on morphological and anatomical features is difficult. To solve this problem isozyme methods were developed. As a result of these studies new cryptic species were recognized and new hypotheses concerning phylogenetic relationship in Pellia were formed. To examine hypotheses derived from isozyme data we decided to study species of the genus Pellia at the DNA level. Total DNA from all Polish Pellia species was isolated. The taxonomic and phylogenetic relationships in Pellia were examined using intergenic spacer sequences between nuclear tRNA(Leu) genes organised in tandem arrays. PCR (polymerase chain reaction) amplification of tRNA(Leu) gene spacers produced fragments of different sizes in all species and their restriction analysis showed species-specific patterns. PCR products were cloned and sequenced. Nucleotide sequence data were used for phylogenetic reconstruction. Sequence analysis confirmed previous results based on isozyme studies that P. endiviifolia- species A and species B as well as P. epiphylla- species S and species N (having different isozyme multilocus genotypes) are separate sibling species. Our results also confirmed the allopolyploid character of the only polyploid species in Pellia, P. borealis which was formed by hybridization of P. epiphylla- species N and P. epiphylla- species S cryptic species.     

Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.     

A phylogenetic analysis of Doronicum (Asteraceae, Senecioneae) based on morphological, nuclear ribosomal (ITS), and chloroplast (trnL-F) evidence.

A phylogenetic analysis of the Old World genus Doronicum (26 species, 4 subspecies) based on sequence data of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, the chloroplast spacer trnL-F, and morphology is presented. Congruence among the three data sets was explored by the computing of several indices, all of which suggest homogeneity between only the two molecular matrices. We argue that the morphological data set contains poor phylogenetic signal and advocate simultaneous analysis of the three data sets (total evidence approach) so that morphological characters are tested for homology by congruence with molecular data. The resulting phylogenetic hypothesis allows several well-supported conclusions including the placement of a Corsican endemic (D. corsicum), sister to the remainder of the genus, and the inference that an early southern European or Mediterranean diversification took place in the genus. Shifts in morphological characters (e.g., homocarpy to heterocarpy) are confirmed to have evolved several times. Results from comparative studies of sequence data of the chloroplast gene ndhF support inclusion of Doronicum in tribe Senecioneae.     

Internal transcribed spacer rDNA can be used to infer the phylogenetic relationships of species within the genus Nematodirus (Nematoda: molineoidea)

Sequences of the first internal transcribed spacer rDNA were characterised for four veterinary important species of gastrointestinal nematodes from the genus Nematodirus. The sequence data were combined with previously published data of the second internal transcribed spacer to determine whether these rDNA regions provided a suitable number of informative characters to determine the phylogenetic relationships of species within the genus. A total of 32 alignment positions of the first internal transcribed spacer data set and 33 characters from the second internal transcribed spacer data set were informative in phylogenetic analyses. Irrespective of whether the data from each spacer were analysed separately or combined, only one most parsimonious tree was produced, with the relationships of the four species fully resolved. In addition, several regions of conservatism in the first internal transcribed spacer sequence among the four Nematodirus species suggests that this rDNA region may also provide phylogenetic information for higher taxonomic levels within the Molineoidea.     

Phylogeny of the Southeast Asian freshwater fish genus Pangio (Cypriniformes; Cobitidae)

The genus Pangio is one of the most species-rich of the loach family Cobitidae and widespread across South and Southeast Asia. Its species diversity has never been studied under a clear phylogenetic approach, but four 'species-groups' were proposed according to the most obvious morphological characters. We present here phylogenetic analyses of the genus Pangio based on sequence data of the mitochondrial cytochrome b gene, the nuclear recombination-activating gene 1 (RAG 1) and a combined dataset of 109 specimens from 18 morphologically identified species across the whole distribution area of the genus. Our data reveal the existence of three major lineages within Pangio. Two of our major lineages were congruent with formerly proposed species-groups, the remaining two species-groups together formed the third major lineage; herein we refer to the lineages as to anguillaris-group, kuhlii-oblonga group and shelfordii-group. The application of a molecular clock dated the age of the three lineages to 33-29 million years. At the species level, our data suggest about 30 distinct lineages, indicating that there is a high number of undescribed species within Pangio. The use of Pangio to address biogeographic questions is demonstrated with the example of the shelfordii-group, which is distributed across Sundaland.     

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.     

Phylogenetic origins of Lophocereus (Cactaceae) and the senita cactus-senita moth pollination mutualism

Recent ecological research has revealed that the Sonoran Desert columnar cactus Lophocereus and the pyralid moth Upiga virescens form an obligate pollination mutualism, a rare but important case of coevolution. To investigate the phylogenetic origins of this unusual pollination system, we used molecular sequence data to reconstruct the phylogeny of the four taxa within the genus Lophocereus and to determine the phylogenetic position of Lophocereus within the North American columnar cacti (tribe Pachycereeae). Our analysis included Lophocereus, six Pachycereus species, Carnegiea gigantea, and Neobuxbaumia tetetzo within the subtribe Pachycereinae, and Stenocereus thurberi as an outgroup within the Stenocereinae. Extensive screening of chloroplast and mitochondrial genomes failed to reveal sequence variation within Lophocereus. At a deeper phylogenetic level, however, we found strong support for the placement of Lophocereus within Pachycereus as sister group to the hummingbird-pollinated P. marginatus. We discuss possible hypotheses that may explain the transition from bat pollination (ancestral) to moth and hummingbird pollination in Lophocereus and P. marginatus, respectively. Additional phylogenetic analyses suggest that the genus Pachycereus should be expanded to include Lophocereus, Carnegiea, Neobuxbaumia, and perhaps other species, whereas P. hollianus may need to be excluded from this clade. Future study will be needed to test taxonomic distinctions within Lophocereus, to test for parallel cladogenesis between phylogroups within Lophocereus and Upiga, and to fully delineate the genus Pachycereus and relationships among genera in the Pachycereinae.     

REEXAMINATION OF PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA): AN ANALYSIS OF atpB AND rbcL GENE SEQUENCES

The chloroplast-encoded atp B gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbc L gene sequence data.1128 base pairs in the coding regions of the atp B gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera— Eudorina, Pleodorina, and Volvox, excluding the section Volvox (= Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbc L gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbc L gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbc L gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atp B gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atp B and rbc L genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.     

Detection of single nucleotide polymorphisms within the Listeria genus using an 'asymmetric' fluorogenic probe set and fluorescence resonance energy transfer based-PCR

AIMS: We describe a novel and inexpensive fluorescence energy transfer (FRET)-based PCR protocol to distinguish single nucleotide polymorphisms (SNPs) within the genus Listeria. METHODS AND RESULTS: Sequence information for the 16S rRNA gene of representative Listeria species was used to design genus-specific primers and two species-specific probes that differed in sequence by one single nucleotide. The probes were 5' labelled with either fluorescein or Texas Red, quenched with a shorter yet complementary 3' dimethyl-amino-phenyloazo benzoic acid (DABCYL) labelled oligonucleotide, and then incorporated into a previously reported 'asymmetric' FRET-based PCR detection protocol. CONCLUSIONS: Listeria monocytogenes could be readily distinguished from other members of the Listeria genus after PCR amplification and measurement of endpoint fluorescence at two different wavelengths. SIGNIFICANCE AND IMPACT OF THE STUDY: The relatively low cost and high flexibility of this system will benefit laboratories in their efforts to develop rapid and specific methods to detect minor sequence differences between related microorganisms.     

Geographical diversification of the genus Cicer (Leguminosae: Papilionoideae) inferred from molecular phylogenetic analyses of chloroplast and nuclear DNA sequences

Cicer L. (Leguminosae: Papilionoideae) consists of 42 species of herbaceous or semi-shrubby annuals and perennials distributed throughout the temperate zones of the Northern Hemisphere. The origin and geographical relationships of the genus are poorly understood. We studied the geographical diversification and phylogenetic relationships of Cicer using DNA sequence data sampled from two plastid regions, trnK / matK and trnS - trnG , and two nuclear regions, the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of nuclear ribosomal DNA, from 30 species. The results from the phylogenetic analyses of combined nuclear and chloroplast sequence data revealed four well-supported geographical groups: a Middle Eastern group, a West-Central Asian group, an Aegean–Mediterranean group, and an African group. Age estimates for Cicer based on methods that do not assume a molecular clock (for example, penalized likelihood) demonstrate that the genus has a Mediterranean origin with considerable diversification in the Miocene/Pliocene epochs. Geological events, such as mountain orogenesis and environmental changes, are major factors for the dispersal of Cicer species. The early divergence of African species and their geographically distinct region in the genus suggest a broader distribution pattern of the genus in the past than at present.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 154 , 175–186.     

New species and unexpected diversity of socially parasitic bees in the genus Inquilina Michener (Hymenoptera: Apoidea: Apidae)

Abstract  Allodapine bees present particular problems for taxonomy due to a high level of morphological conservatism in adults, even between genera. However, this tribe of bees also presents a unique opportunity to explore the evolution of social parasitism because of the comparatively large number of origins of socially parasitic species. Morphological differences presented here, along with DNA sequence data and molecular phylogenetic analyses, indicate a much larger number of Australian social parasite species in the genus Inquilina than previously anticipated, and suggest that the final number of socially parasitic species may be considerable. We describe five new species and present sequence data that will help elucidate the delineation of further new species. Inquilina provides a unique opportunity to study the evolution of social parasitism in social insects, but further studies will need to encompass both population genetic and phylogenetic approaches.     

Molecular phylogenetics and diversification of the genus Sporophila (Aves: Passeriformes)

The evolutionary affinities within and among many groups of nine-primaried oscines remain unresolved. One such group is Sporophila, a large genus of New World tanager-finches. Our study focused particularly on clarifying the relationship between this genus and a closely related one, Oryzoborus, and on examining the phylogenetic affinities of the "capuchinos," a group of 11 Sporophila species that share a similar male plumage coloration pattern. Our phylogenetic analyses, based on 498 bp of mitochondrial DNA sequence, indicated that: (1) Oryzoborus is embedded within a well-supported clade containing all Sporophila species, which strongly suggests that both genera should be merged, (2) the species of capuchinos comprise a monophyletic group, implying that the plumage patterns common to all probably arose only once, and (3) the capuchinos clade is comprised of two sub-clades, one including two species that are distributed in northern South America and the other one containing eight species that are present south of the Amazon River. Mean sequence divergence among the southern capuchinos species was extremely low, suggesting a rapid radiation within the last half-million years that may be related to the high level of sexual selection present in the genus and might have been promoted by marine ingressions and egressions that occurred in some southern coastal regions of South America in the Late Pleistocene.     

The origin and evolution of seahorses (genus Hippocampus): a phylogenetic study using the cytochrome b gene of mitochondrial DNA

Phylogenetic relationships among 93 specimens of 22 species of seahorses (genus Hippocampus) from the Atlantic and Indo-Pacific Oceans were analysed using cytochrome b gene sequence data. A maximum sequence divergence of 23.2% (Kimura 2-parameter model) suggests a pre-Tethyan origin for the genus. Despite a greater number of seahorse species in the Indo-Pacific than in the Atlantic Ocean, there was no compelling genetic evidence to support an Indo-Pacific origin for the genus Hippocampus. The phylogenetic data suggest that high diversity in the Indo-Pacific results from speciation events dating from the Pleistocene to the Miocene, or earlier. Both vicariance and dispersal events in structuring the current global distribution of seahorses. The results suggested that several species designations need re-evaluating, and further phylogeographic studies are required to determine patterns and processes of seahorse dispersal.