Genetic variability of trypanosomatids isolated from phytophagous hemiptera defined by morphological, biochemical, and molecular taxonomic markers.

In the present study, we investigated the genetic variability among 49 new isolates of trypanosomatids from phytophagous Hemiptera by means of morphological characters, growth features, and biochemical (enzymes of ornithine-arginine cycle) and molecular markers (based on spliced-leader, and ribosomal genes). From 402 phytophagous insects dissected and examined for the presence of trypanosomatids, 228 species belonging to Pyrrhocoridae, Coreidae, Lygaeidae, and Pentatomidae families harbored trypanosomatids in their salivary glands, or digestive tubes. Among these insects, 211 carried promastigotes and only 17 had choanomastigote forms. The results show a strong association among morphology, growth features, and biochemical and molecular markers and reveal the genetic diversity of the isolates, which were assigned to Crithidia, Phytomonas, and Leptomonas; we found genetic polymorphism within all these genera, thus indicating high genetic variability among trypanosomatids from phytophagous insects.     

Ultrastructural and molecular analyses of Rhizophydiales (Chytridiomycota) isolates from North America and Argentina

The Rhizophydiales is the most recently circumscribed order in the Chytridiomycota. Past studies focused on soil chytrids from North America and Australia to determine the range of diversity within this clade of chytrids and established three families (Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae) in the new order. Although Rhizophydiales contains seemingly simple chytrids morphologically, analyses of ribosomal gene sequences and zoospore characters have demonstrated unexpected genetic and ultrastructural diversity, highlighting the need for broader habitat and geographic sampling to reveal the actual diversity within this new order. To enlarge our sampling, in this study we investigated 38 newly cultured chytrids collected from aquatic habitats in Argentina, a territory under-explored for chytrid diversity. From analyses of thallus morphology, zoospore ultrastructure, and 28S and ITS1–5.8S–ITS2 ribosomal gene sequences, we expand the concept of Rhizophydiales, describing seven new families (Alphamycetaceae, Angulomycetaceae, Aquamycetaceae, Globomycetaceae, Gorgonomycetaceae, Pateramycetaceae, and Protrudomycetaceae) and eight new genera (Alphamyces, Angulomyces, Aquamyces, Globomyces, Urceomyces, Gorgonomyces, Pateramyces, and Protrudomyces). Results of this study underscore that even more extensive sampling from varied habitats and geographies is needed to adequately ascertain the diversity of chytrids in the Rhizophydiales.     

Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters

A set of 24 strains belonging to the genus Anabaena (Phylum Cyanobacteria), isolated from diverse geographic locations in India, were evaluated along with three International type strains of Anabaena (ATCC 29414, ATCC 29208 and ATCC 27899) for their morphological, physiological and biochemical diversity. The morphological dataset, consisting of 58 variants for 15 characters, and SDS-PAGE protein profiles comprising 17 polymorphic bands were utilized to differentiate the selected Anabaena strains and explore the patterns of diversity through cluster analysis. Physiological and biochemical characterization with respect to nitrogen fixation and accumulation of chlorophyll and phycobiliproteins led to the identification of some highly promising Anabaena strains for use as biofertilizers and source of pigments. The study highlighted the tremendous inter and intraspecific diversity within the Anabaena isolates and indicated the potential as well as constraints of the morphological and protein profiling datasets for unambiguous differentiation and analyses of diversity among the Anabaena strains.     

Genetic relationships among ten endod types as revealed by a combination of morphological,RAPD and AFLP markers

The genetic relationships among ten types of endod (Phytolacca dodecandra) cultivated by the Institute of Pathobiology of the Addis Ababa University to combat the disease bilharzia in Ethiopia were studied using morphology and molecular markers. A total of 18 morphological characters, 194 amplified fragment length polymorphism (AFLP) and 42 random amplified polymorphic DNA (RAPD) markers were used to determine genetic proximity between types. Genetic distance and cluster analysis of the AFLP data revealed the lack of genetic difference between E47 and E48 but relatively wider genetic difference among the other endod types. Cluster and principal component analyses performed on the AFLP and RAPD markers demonstrated the presence of distinct separation of E56 but not that of E44 from the others. The AFLP and RAPD data, thcrefore, did not support the hypothesis that the superiority of E44 in agronomic traits and molluscicidal potency is linked to its distinct genetic difference from the other endod types. Matrices correspondence tests demonstrated the presence of greater correspondence between AFLP and RAPD data (r = 0.842) but not between the morphology and that of AFLP and RAPD. This indicates the correspondence more between the two DNA markers systems than either of them with morphological traits. The cophenetic correlation coefficients also revealed poor fit for morphology (r = 0.716), good fit for RAPD (r = 0.872) and very good fit for AFLP (r = 0.975), reflecting the hyper-variability and higher resolving power of AFLP.     

Characteristics of the energy-transducing NADH-quinone oxidoreductase ofParacoccus denitrificans as revealed by biochemical,biophysical, and molecular biological approaches

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.     

Population genetic structure and trait associations in forest savory using molecular,morphological and phytochemical markers

In this investigation, morphological, phytochemical and ISSR markers were used to estimate the relationships among and within seven populations of white savory (Satureja mutica), belonging to four provinces in Iran. The individuals were phenotypically diverse, which stamen length, corolla length, corolla diameter, calyx length, bract length, inflorescence length, calyx length and bracteole width were characteristics with the highest variation. Leaf dimensions were in significant correlation with flower and inflorescence characteristics. Chemical compounds of essential oils were found variable in various individuals and all samples were principally composed of phenolic constituents (carvacrol and/or thymol). As a consequence, the plants were classified into two major chemotypes including carvacrol and thymol. A total of 197 band positions were produced by 14 ISSR primers, of which 176 were found polymorphic with 88.91% polymorphism. ISSR genetic similarity values among individuals ranged between 0.45 and 0.94 which was indicative of a high level of genetic variation. Multiple regression analysis (MRA) revealed that phytochemical compositions as dependent variable, showed statistically significant correlation and in association with leaf and flower traits as independent variable, indicating a main role of leaf and flower on production of these compounds. Also, several ISSR fragments were found associated with some morphological traits and phytochemical compositions. The high diversity within and among populations of S. mutica according to different data systems could provide useful information for conservation and selection of cross-parents in breeding programs.     

Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

AIMS: Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification. METHODS AND RESULTS: Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus.     

Lophodermium pini-mugonis sp. nov. on needles of Pinus mugo from the Alps based on morphological and molecular data

Lophodermium pini-mugonis, collected on needles of Pinus mugo from German Alps, is described as a species new to science. It is characterized by subcuticular ascomata with a wrinkled surface and a somewhat untidy outline, a complex structure of lip cells, and ellipsoidal conidia. An analysis of the internal transcribed spaces of rDNA showed that Lophodermium pini-mugonis is, sister to Lophodermium autumnale and distantly related to other Lophodermium species on pines. The hypothesis of cospeciation of Lophodermium species with members of the Pinaceae is discussed.     

A new species of Fomitiporia (Hymenochaetaceae, Basidiomycota) from China based on morphological and molecular characters

Fomitiporia punicata sp. nov., collected from Shaanxi Province, northern China, and so far exclusively on Punica granatum (Punicaceae), is described and illustrated, and its preliminary phylogenetic relationships are inferred based on sequence data from the ribosomal ITS-5.8S regions. Fomitiporia punicata belongs to the F. robusta–F. punctata complex, but is characterized by an effused–reflexed to pileate basidioma with triquetrous to ungulate pilei, pale yellowish brown to cinnamon brown pore surface, moderately thick-walled skeletal hyphae in the context, which are partly collapsed on drying, lack of setae, presence of fusoid to subulate cystidioles, and growth on Punica. It also forms a distinct clade within Fomitiporia, and is related to some species from the temperate or Mediterranean zone of the northern hemisphere. An identification key to the species of Fomitiporia recorded in China is provided. Fomitiporia tibetica is transferred to Pseudoinonotus.     

Fingerprinting of freshly separated and cultured cyanobionts from different Azolla species using morphological and molecular markers

Differentiation of freshly separated (uncultured) and cultured cyanobionts of Azolla species was carried out employing morphological markers and profiles generated using SDS-PAGE and PCR–RFLP of 16S rRNA. The cell dimensions of vegetative cells, heterocysts and heterocyst frequency (25–28%) of the freshly separated cyanobionts were distinctly higher than that those recorded for the cultured cyanobionts (7–10%). The SDS-PAGE profiles of whole cell proteins of cultured cyanobionts (comprising 28–30 bands) and those of freshly separated cyanobionts (consisting of 6–10 bands) exhibited distinct differences and unique bands. AluI was able to discriminate freshly separated cyanobionts from cultured cyanobionts of same species of Azolla. The profiles of cyanobionts (freshly separated and cultured) of A. rubra (RU6503) generated using the restriction enzyme AluI were distinctly different from other cyanobionts and unique bands were observed in both cyanobionts. The cultured cyanobionts from A. microphylla (MI4018), A. filiculoides (FI1001) and A. pinnata (PP7001) showed the presence of a distinct band of 450, 622 and 307 bp, respectively. Three common bands of 500, 400 and 275 bp were recorded in the AluI restriction profiles of all the freshly separated cyanobionts. 16S rRNA PCR–RFLP analyses confirmed the existence of primary and secondary cyanobionts in the leaf cavities of different Azolla species. These techniques can be utilized for discriminating between freshly separated and cultured cyanobionts of Azolla and provide reliable fingerprints for these strains.     

Morphological and molecular characterization of Pisolithus occurring in Hokkaido Island, Northern Japan

Pisolithus basidiomes were found under different forest trees in Hokkaido Island, Japan. These basidiomes were characterized morphologically and molecularly. Although presenting different basidiome morphology and growing under different hosts, specimens presented similar spores ornamentation, and diameters. These spores had coarse, crowded, and blunted spines with three to eight basidiospores per basidium. Ribosomal DNA-based phylogenetic analysis indicated that variability of Pisolithus in this area is low. Phylogenetic analysis showed that Pisolithus analyzed in this study did not group with Pisolithus specimens from other geographical origins. These results suggest that Pisolithus from this area should be taxonomically distinguished from other Pisolithus.     

Molecular markers reveal differentiation among isolates of Coccidioides immitis from California, Arizona and Texas

Coccidioides immitis causes coccidioidomycosis, a fungal disease of both immunocompromised and otherwise healthy people; it is capable of causing large epidemics and the disease is often refractory to chemotherapy. To quantify the magnitude of population differentiation and estimate levels of gene flow in C. immitis , multilocus genotypes were scored for 20–25 clinical isolates from each of Bakersfield (California), Tucson (Arizona), and San Antonio (Texas). The molecular markers used were PCR products with polymorphic restriction endonuclease sites, found and characterized in a previous study of the Tucson population. The data show very highly significant differences in allele frequencies between all three populations, and suggest very low levels of migration between populations. One isolate in the San Antonio sample was an outlier, showing the California-specific allele at all four of the loci distinguishing the two populations, and subsequent inquiries indicated that the infection had indeed been acquired in California. Thus, genetic information can be used to infer the geographical origin of a fungal infection.     

Some morphometric,anatomical and biochemical characteristics of fruits and seeds of Onobrychis spp. in Italy

ABSTRACT

The fruits, seed coats and seed storage proteins of Onobrychis arenaria, O. montana, O. viciifolia, O. alba, O. supina and O. caput-galli were studied to examine the variability between and within species. The morphometric characteristics of the fruit had a high intraspecific variability which often exceeded that between species. Only the fruit of O. caput-galli had values for length, width, thickness and number of thorns which were almost always higher than those of the other species. The anatomical characteristics of the seed coats were extremely variable. The highest values of seed coat thickness were recorded in the diploid species. The palisade-like layer (or Malpighian cells) in O. caput-galli differed in size and morphology from that of the other species. The electrophoretic analysis revealed that the number and position of storage proteins varied between and within species.     

Phenetic investigation of non-nodulating African species ofAcacia (Leguminosae) using morphological and molecular markers

Morphological and RAPD markers were used to assess the relationships among nodulating and non-nodulating species of AfricanAcacia. Non-nodulating species of AfricanAcacia are only found within subg.Aculeiferum sect.Monacanthea. African species of sect.Monacanthea examined were found to form a group distinct from the other African species examined on a morphological and molecular basis. All lack the ability to nodulate, suggesting that non-nodulation may be used as a taxonomic tool. The species of sect.Aculeiferum were separated by RAPD and morphological analysis into two groups depending on whether they were armed with prickles in pairs and/or prickles in threes, or solitary. A third group of species was identified within sect.Acacia: further subdivision of this group was achieved into subsectt.Pluriseriae andUniseriae. The position ofA. albida relative to other AfricanAcacia species was found to be distinct but not totally independent of the genus. The partitioning and distribution of the genetic variability within the genus is further elucidated by the RAPD analysis of populations ofAcacia species. A population analysis ofA. polyacantha demonstrated geographical and site-specific variation.     

Eight new species of the spider genera Raveniola and Sinopesa from China and Vietnam (Araneae,Nemesiidae)

Eight new species, seven Raveniola Zonstein, 1987 and one Sinopesa Raven & Schwendinger, 1995 from China and Vietnam are described: Raveniolaalpinasp. n., Raveniolabellulasp. n., Raveniolachayisp. n., Raveniolagracilissp. n., Raveniolarugosasp. n., Raveniolaspirulasp. n. and Raveniolayajiangensissp. n. and Sinopesaninhbinhensissp. n. Keys to all East-Asian congeners, diagnoses of the new species, and new distribution data of Raveniolamontana Zonstein & Marusik, 2012, with a first record for Sichuan, China, are provided.     

Phylogeny of the Baldratiina (Diptera: Cecidomyiidae) inferred from morphological, ecological and molecular data sources, and evolutionary patterns in plant-galler relationships

The phylogeny of the gall-midge subtribe Baldratiina (Diptera: Cecidomyiidae) was reconstructed from molecular (partial sequence of the mitochondrial 12S rDNA), morphological and ecological data sets, using 16 representative species of most of the genera. The morphological and ecological data were combined in a single character matrix and analyzed separately from the molecular data, resulting in an eco-morphological cladogram and a molecular cladogram. Attributes of galls and host associations were superimposed on the molecular cladogram in order to detect possible trends in the evolution of these traits. The cladograms resulting from the two independent analyses were statistically incongruent, although both provide evidence for the monophyly of the genera Baldratia and Careopalpis and the paraphyly of the genera Stefaniola and Izeniola. The results suggest a minor impact of the morphological characters traditionally used in the classification of the Baldratiina, whereas ecological data had a major impact on the phylogenetic inference. Mapping of gall and host attributes on the molecular cladogram suggests that multi-chambered stem galls constitute the ancestral state in the subtribe, with several subsequent shifts to leaf galls. It is concluded that in contrast to other studied groups of gall insects, related baldratiine species induce different types of galls, attesting to speciation driven by gall-type shifts at least as often as host shifts.     

Genetic variation among natural isolates of the ectomycorrhizal hypogenous fungus,Rhizopogon roseolus from Japanese pine forests inferred using AFLP markers

Genetic variation among 45 Rhizopogon roseolus isolates from 21 different regions of Japan were inferred using amplified fragment length polymorphism (AFLP) markers. Using three primer pair combinations, AFLP analysis reproducibly produced a total of 223 DNA fragments, 74.4% of which were polymorphic. Pairwise dissimilarity of AFLP patterns between isolates ranged from 0.043 to 0.228. Cluster analysis and principal coordinate analysis of AFLP data generally showed four major clusters from geographically distinct areas. The findings suggested that the Japanese populations of R. roseolus from different geographical regions can be distinguished based on AFLP characters.     

Speciation pattern of Telestes souffia complex (Teleostei,Cyprinidae) in Europe using morphological and molecular markers*

Gilles, A., Costedoat, C., Barascud, B., Voisin, A., Banarescu, P., Bianco, P. G., Economidis, P. S., Mari?, D. & Chappaz, R. (2010). Speciation pattern of Telestes souffia complex (Teleostei, Cyprinidae) in Europe using morphological and molecular markers.—Zoologica Scripta, 39, 225–242. It is notorious that many species boundaries are erroneously defined. When molecular markers are used, misleading evidence can notably be due to the characteristics inherent to mitochondrial DNA and quantity of markers used but also because of a limited range distribution sampling. European cyprinids biodiversity inventory is still an ongoing task and surprising phylogeographic patterns and phylogenetic relationships are still recovered on account of methodological evolution. This is particularly obvious for the Telestes souffia complex. This species occurs in a fragmented range and species boundaries is greatly debated. In this study, we provide an updated delimitation of the different evolutionary entities constituting this T. souffia complex and propose a taxonomic revision. Morphological and molecular analyses were carried out on 520 specimens coming from 19 localities representing the complete geographical range of this species complex and six related sister species that could potentially interact with it (outgroup ‘sensu lato’). Phylogenetic reconstructions and multivariate analyses demonstrated that the T. souffia complex is constituted of at least three species (T. souffia, Telestes muticellus and Telestes montenigrinus). Data also suggested that T. souffia comprises three subspecies (T. s. souffia, T. s. agassii; T. s. rysela). We also confirm the splitting of the T. souffia sister species Telestes pleurobipunctatus into two distinct species. The Peloponnesian lineage will be referred as Telestes alfiensis and the continental lineage as T. pleurobipunctatus. Morphological and molecular markers displayed some degree of incongruence within T. souffia suggesting that introgressive hybridization has played a role in the evolution of the Telestes genus. However, discordance among data sets could also result from heterogeneous rate of morphological evolution. Finally, we demonstrated that T. muticellus was implicated in two categories of hybridization: an inter‐species hybridization (between T. muticellus and T. souffia) and an inter‐generic hybridization (between T. muticellus and Squalius lucumonis), a phenomenon rarely observed for a same species.