Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

Background: Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method. Results: We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP. Conclusions: Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.

HYBRID, SUBSPECIES, OR SPECIES? THE VALIDITY AND TAXONOMIC STATUS OF PHAETHORNIS LONGUEMAREUS AETHOPYGA ZIMMER, 1950 (TROCHILIDAE)

Phaethornis longuemareus aethopyga was described by John T. Zimmer in 1950 and treated as a valid subspecies until it was proposed that the three known specimens were hybrids between R ruber and P. rupurumii amazonicus. On the basis of some recently collected specimens, we reevaluated the validity of P. l. aethopyga. Despite showing some differences related to age and sex, all specimens agree in the general plumage pattern and are fully diagnosable when compared with any other taxon of the genus. The hypothesis of a hybrid origin becomes unsustainable when one notes that (1) P. l. aethopyga has characters that are unique and absent in the purported parental species, such as the white outer margins at the base of the rectrices; and (2) P. l. aethopyga occurs far from the distribution of one of the alleged parental species. Furthermore, field data show that P. l. aethopyga has attributes typical of a valid and independent taxon, such as lekking behavior. Therefore, given its overall diagnosis, P. aethopyga could at least be treated as a phylogenetic species. Yet its morphological and vocal distinctiveness with respect to other Phaethornis spp. in the ""Pygmornis group"" is greater than that observed between some species pairs traditionally regarded as separate biological species within the group, which supports its recognition as a species under the biological species concept. Received 13 July 2008, accepted 9 March 2009.

PLANKTON-CAUGHT ZOEAL STAGES AND MEGALOPA OF THE LOBSTER SHRIMP AXIUS SERRATUS (DECAPODA: AXIIDAE) FROM THE BAY OF FUNDY, CANADA, WITH A SUMMARY OF AXIIDEAN AND GEBIIDEAN LITERATURE ON LARVAL DESCRIPTIONS

Wild-caught larvae, attributed to the lobster shrimp Arius serratus, consisting of two zoeal stages and a decapodid (megalopa), are described in detail. Parentage of larvae was ascertained based on geographic distribution of axiideans and gebiideans (= former thalassinideans) within the study area and close morphological resemblance to other congeneric larval stages. Larvae of A. serratus represent the first described 'thalassinidean' larvae from Canadian Atlantic waters and the first for Axiidae within the northwest Atlantic. Among axiidean larvae, those of A. serratus most closely resemble larvae of A. stirhynchus from the eastern Atlantic. Distinct features include the spination of the pleon that set A. serratus zoeae apart from those of most other 'thalassinideans' but that, in combination with a telson very similar to Homarus americanus, contributes to the general resemblance of A. serratus larvae to those of the American lobster. The primary distinction between these taxa is the presence of a chela on the third pereiopod in the latter that is not present in the former. In view of these appendages being prone to loss or damage, other characters that separate these taxa are listed and discussed. Given the uncertain status of some taxa within Axiidae and limited detailed information of larvae with certain parentage, difficulties in delineating the family based on larvae persist, as they do for cladistic analyses using adult morphology and molecular approaches.

Phylogeny and Taxonomy of the Neotropical Thepytus (Lepidoptera: Lycaenidae: Theclinae)

The Neotropical lycaenid hairstreak genus Thepytus Robbins and its eight species are revised. Species treatments summarize nomenclature, distribution, habitat, behavior, and diagnostic traits, as well as noting why each species is considered distinct under a biological species concept. An identification key for males and a checklist are included. Beatheclus Balint & Dahners new synonym is synonymized with Thepytus, and Theppus beatrizae (Balint & Dahners) is a new combination. Other nomenclatural actions include the description of Thepytus jennifer Busby & Robbins new species. Thepytus nancyana Busby & Robbins new species, and Thepytus carmen Robbins & Duarte new species. A lectotype is designated or Thecla thyrea Hewitson, 1867, to ensure stability of this name. A phylogenetic analysis based on 22 coded morphological characters yields one equal weight most parsimonious 39-step tree. Implied weighting does not change the tree topology. Unambiguous changes in elevation optimized on the cladogram show that a montane lineage of Thepytus colonized the lowlands in at least one instance. The use of T. echelta (Hewitson) as a biological control agent for Psittacanthus (Loranthaceae) is discussed.

Molecular and morphological characterization of Amathia distans Busk and Amathia brasiliensis Busk (Bryozoa: Ctenostomata) from the tropical and subtropical Western Atlantic

Morphological and molecular analyses have proven to be complementary tools of taxonomic information for the redescription of the ctenostome bryozoans Amathia brasiliensis Busk, 1886 and Amathia distans Busk, 1886. The two species, originally described from material collected by the `Challenger` expedition but synonymized by later authors, now have their status fixed by means of the selection of lectotypes, morphological observations and analyses of DNA sequences described here. The morphological characters allowing the identification of living and/or preserved specimens are (1) A. brasiliensis: whitish-pale pigment spots in the frontal surface of stolons and zooids, and a wide stolon with biserial zooid clusters growing in clockwise and anti-clockwise spirals along it, the spirality direction being maintained from maternal to daughter stolons; and (2) A. distans: bright yellow pigment spots in stolonal and zooidal surfaces including lophophores, and a slender stolon, thickly cuticularized, with biserial zooid clusters growing in clockwise and anti-clockwise spirals along it and the spirality direction not maintained from maternal to daughter stolons. Pairwise comparisons of DNA sequences of the mitochondrial genes cytochrome c oxidase subunit I and large ribosomal RNA subunit revealed deep genetic divergence between A. brasiliensis and A. distans. Finally, analyses of those sequences within a Bayesian phylogenetic context recovered their genealogical species status.

Individual-level diet variation in four species of Brazilian frogs

Many natural populations exploiting a wide range of resources are actually composed of relatively specialized individuals. This interindividual variation is thought to be a consequence of the invasion of `empty` niches in depauperate communities, generally in temperate regions. If individual niches are constrained by functional trade-offs, the expansion of the population niche is only achieved by an increase in interindividual variation, consistent with the `niche variation hypothesis`. According to this hypothesis, we should not expect interindividual variation in species belonging to highly diverse, packed communities. In the present study, we measured the degree of interindividual diet variation in four species of frogs of the highly diverse Brazilian Cerrado, using both gut contents and delta(13)C stable isotopes. We found evidence of significant diet variation in the four species, indicating that this phenomenon is not restricted to depauperate communities in temperate regions. The lack of correlations between the frogs` morphology and diet indicate that trade-offs do not depend on the morphological characters measured here and are probably not biomechanical. The nature of the trade-offs remains unknown, but are likely to be cognitive or physiological. Finally, we found a positive correlation between the population niche width and the degree of diet variation, but a null model showed that this correlation can be generated by individuals sampling randomly from a common set of resources. Therefore, albeit consistent with, our results cannot be taken as evidence in favour of the niche variation hypothesis.

Morphological Studies on the Mature Worker Larvae of Paratrechina fulva (Hymenoptera, Formicidae)

The ant Paratrechina fulva is native to Brazil, but has spread to other countries where it has become a serious pest. Their larvae are known from a preliminary description made without the aid of scanning electron microscopy. The present investigation aimed at describing the mature worker larvae of this species in deeper detail, while identifying useful morphological characters for taxonomy and cladistics. General morphological traits typical of Paratrechina larvae described so far were also confirmed with this species, particularly regarding body shape, types of hairs, and distribution pattern of head sensilla, but P fulva presented unusually shaped mandibles.

A NOVEL EPIPHYTIC CYANOBACTERIAL SPECIES FROM THE GENUS BRASILONEMA CAUSING DAMAGE TO EUCALYPTUS LEAVES

A cyanobacterial mat colonizing the leaves of Eucalyptus grandis was determined to be responsible for serious damage affecting the growth and development of whole plants under the clonal hybrid nursery conditions. The dominant cyanobacterial species was isolated in BG-11 medium lacking a source of combined nitrogen and identified by cell morphology characters and molecular phylogenetic analysis (16S rRNA gene and cpcBA-IGS sequences). The isolated strain represents a novel species of the genus Brasilonema and is designated Brasilonema octagenarum strain UFV-E1. Thin sections of E. grandis leaves analyzed by light and electron microscopy showed that the B. octagenarum UFV-E1 filaments penetrate into the leaf mesophyll. The depth of infection and the mechanism by which the cyanobacterium invades leaf tissue were not determined. A major consequence of colonization by this cyanobacterium is a reduction in photosynthesis in the host since the cyanobacterial mats decrease the amount of light incident on leaf surfaces. Moreover, the cyanobacteria also interfere with stomatal gas exchange, decreasing CO2 assimilation. To our knowledge, this is the first report of an epiphytic cyanobacterial species causing damage to E. grandis leaves.

Notes on Acaulospora bireticulata Rothwell & Trappe and first record of Acaulospora koskei Blask. from Brazil)

(Notes on Acaulospora bireticulata Rothwell & Trappe and first record of Acaulospora koskei Blask. from Brazil). Our knowledge of species distribution in arbuscular mycorrhizal fungi (AMF) is still limited and morphological variations found in isolates of one species may provide additional insight for understanding the characters used in the taxonomy of this group. The aims of this work were to expand biogeographical data regarding Acaulospora koskei Blaszk. and Acaulospora bireticulata Rothwell & Trappe, both found in an Araucaria Forest in Sao Paulo state, Brazil, as well as to compare the descriptions of these species with those of other similar AMF.

Description of the Immatures of Workers of the Ant Linepithema micans Forel (Hymenoptera: Formicidae)

Linepithema micans Forel is a poorly studied species that is now considered a pest in vineyards in South Brazil. Larval characters have been used in a few phylogenetic studies, and their importance greatly depends in the amount of available information on different species. This study presents a complete panorama on the external morphology of the immatures of L. micans based on observations by light and scanning electron microscopy. The number of larval instars was estimated as three based on the frequency distribution of head widths of 965 larvae. Larvae of L. micans were similar to other Linepithema Mayr in the general format of body and mandibles, presenting a dorsal abdominal protuberance, nine pairs of spiracle, and unbranched hairs. On the other hand, L. micans was unique for having shorter hairs, predominantly denticulate, intraspecific variation in the number of antennal sensilla and in the types of sensilla on the labial palps were reported. Microsc. Res. Tech. 74:337-342, 2011. (C) 2010 Wiley-Liss, Inc.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Development of novel microsatellites from Moniliophthora perniciosa, causal agent of the witches` broom disease of Theobroma cacao

Moniliophthora perniciosa is the causal agent of the witches` broom disease of cacao. Based on available genomic sequences, we identified 30 new microsatellite loci, which were analysed using 50 isolates from four populations sampled over a wide geographical area in Brazil, including three populations from the Amazon, the fungal putative centre of diversity, plus one from Bahia. Nine loci were polymorphic, with an average of 2.9 alleles per locus. The level of polymorphism observed was low, but these markers may allow the evaluation of pathogen diversity and the establishment of molecular standards for isolate fingerprinting to support cacao breeding.

Floral development in the tribe Cedreleae (Meliaceae, sub-family Swietenioideae): Cedrela and Toona

Background and Aims Floral development of Cedrela and Toona, the genera comprising the basal tribe Cedreleae of the sub-family Swietenioideae of Meliaceae, is described. The focus was on three endangered, ecologically and economically important species: Cedrela fissilis, Cedrela odorata and Toona ciliata. The aims of the study were to characterize the patterns of floral development in the tribe and to establish apomorphic and plesiomorphic floral characters in relation to other taxa within the family based on the current molecular phylogeny of Meliaceae. Methods A detailed floral structural and developmental study was completed using both scanning electron microscopy and visualization of microtome sections with a light microscope. Key Results Twelve floral developmental stages were identified. The initial development of the pentamerous flowers of both Toona and Cedrela is strikingly similar. The morphological differences observed between them are due to differential patterns of organ elongation and adnation/connation occurring late in development. Additionally, the formation of functionally male and female flowers was found to occur at specific positions within the inflorescence. Conclusions Due to the basal position of the tribe Cedreleae in the phylogeny of Meliaceae, functionally either male or female pentamerous flowers and the presence of (at least partially) free stamens may be considered plesiomorphic traits within the family. In contrast, sympetaly and the absence of nectaries in Cedrela species are synapomorphies.

Microsatellite Polymorphisms in Cassava Landraces from the Cerrado Biome, Mato Grosso do Sul, Brazil

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region`s high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.

Systematic review and cladistic analysis of the genus Eusarcus Perty 1833 (Arachnida, Opiliones, Gonyleptidae)

Eusarcus Perty 1833 is one of the oldest described genera of Pachylinae, comprising 36 species distributed from northeastern to southern Brazil (including the central west region), northeastern Argentina, eastern Paraguay and Uruguay. The genus is reviewed and a new classification is proposed based on a cladistic analysis. A cladistic analysis was performed with the 34 valid species of Eusarcus and 11 species belonging to certain Gonyleptidae subfamilies. The data matrix has 67 characters: 14 from dorsal scutum and pedipalp, 38 from male legs and 15 from male genitalia. Two equally parsimonious trees were found (L=319; C. I.=0.26, R. I.=0.61). Pygophalangodus gemignanii uruguayensis Ringuelet 1955a and Pygophalangodus gemignanii gemignanii Mello-Leitao 1931b are here elevated to the category of species, and the following new combinations are proposed: E. catharinensis (Mello-Leitao 1927); E. berlae (Mello-Leitao 1932); E. gemignanii (Mello-Leitao 1931b); E. signatus(Roewer 1949); E. sooretamae (Soares & Soares 1946a); E. uruguayensis (Ringuelet 1955a). The following generic synonymies are proposed: Eusarcus Perty 1833 (type species E. armatus Perty 1833) = Metagraphinotus Mello-Leitao 1927 (type species M. catharinensis Mello-Leitao 1927), Pareusarcus Roewer 1929 (type species P. corniculatus Roewer 1929), Pygophalangodus Mello-Leitao 1931b (type species P. gemignanii-gemignanii Mello-Leitao 1931b) and Antetriceras Roewer 1949 (type species A. signatus Roewer 1949). The following specific synonymies are proposed: Eusarcus hastatus Sorensen 1884 = Pucrolioides argentina Roewer 1913, E. guimaraensi H. Soares 1945, Jacarepaguana pectinifemur Piza 1943, Canestrinia canalsi Mello-Leitao 1931a, and E. maquinensis H. Soares 1966b; E. armatus Perty 1833 = E. curvispinosus Mello-Leitao 1923b, and Enantiocentron montis Mello-Leitao 1936; Eusarcus catharinensis (Mello-Leitao 1927) = E. antoninae Mello-Leitao 1936, E. perpusillus Mello-Leitao 1945, E. tripos Mello-Leitao 1940, and Metagraphinotus trochanterspinosus Soares & Soares 1947b; E. nigrimaculatus Mello-Leitao 1924 = Pareusarcus centromelos Mello-Leitao 1935a, E. furcatus Roewer 1929, Orguesia armata Roewer 1913, and Pareusarcus corniculatus Roewer 1929; E. oxyacanthus Kollar in Koch 1839a = Enantiocentron doriphorus Mello-Leitao 1932, and E. spinimanu Mello-Leitao 1932; E. pusillus Sorensen 1884 = E. vervloeti B. Soares 1944c; E. berlae Mello-Leitao 1932 = Metagraphinotus arlei Mello-Leitao 1935a. Metapucrolia armata (Sorensen 1895) is revalidated, transferred to Eusarcus and considered as a species inquirenda. A new name, Eusarcus metapucrolia is proposed for this species to avoid homonymy with the type species of Eusarcus, E. armatus Perty 1833. Eusarcus aberrans Mello-Leitao 1939a is considered as a species inquirenda. The male of E. teresincola Soares & Soares 1946a is described. Female of the following species are described: E. bifidus Roewer 1929; E. dubius B. Soares 1943b; E. insperatus B. Soares 1944a; E. schubarti Soares & Soares 1946a; E. sooretamae (Soares & Soares 1946a). The following new species are described from Brazil: E. acrophthalmus (type locality: Bahia, Ilheus, Parataquice); E. alpinus (Rio de Janeiro, Santa Maria Madalena, Parque Estadual do Desengano); E. caparaoensis (Minas Gerais, Alto Caparao, Parque Nacional do Caparao); E. cavernicola (Goias, Sao Domingos, Parque Estadual de Terra Ronca, Lapa da Angelica); E. didactylus (Rio de Janeiro, Teresopolis, Parque Nacional Serra dos Orgaos); E. garibaldiae (Santa Catarina, Itajai); E. geometricus (Rio de Janeiro, Teresopolis, Parque Nacional Serra dos Orgaos); E. manero (Rio de Janeiro, Marica, Itaipuacu); E. matogrossensis (Mato Grosso, Chapada dos Guimaraes); E. mirabilis (Minas Gerais, Marlieria, Parque Estadual Rio Doce); E. sergipanus (Sergipe, Itabaiana, Parque Nacional de Itabaiana) and E. tripectinatus (Minas Gerais, Rio Preto). The holotype of E. curvispinosus is proposed as the neotype of E. armatus Perty 1833, the type material of which has been lost. Lectotypes for the following species were designated: E. aduncus; E. hastatus; E. oxyacanthus.