A Multilocus Species Delimitation Reveals a Striking Number of Species of Coralline Algae Forming Maerl in the OSPAR Maritime Area

Maerl beds are sensitive biogenic habitats built by an accumulation of loose-lying, non-geniculate coralline algae. While these habitats are considered hot-spots of marine biodiversity, the number and distribution of maerl-forming species is uncertain because homoplasy and plasticity of morphological characters are common. As a result, species discrimination based on morphological features is notoriously challenging, making these coralline algae the ideal candidates for a DNA barcoding study. Here, mitochondrial (COI-5P DNA barcode fragment) and plastidial (psbA gene) sequence data were used in a two-step approach to delimit species in 224 collections of maerl sampled from Svalbard (78°96’N) to the Canary Islands (28°64’N) that represented 10 morphospecies from four genera and two families. First, the COI-5P dataset was analyzed with two methods based on distinct criteria (ABGD and GMYC) to delineate 16 primary species hypotheses (PSHs) arranged into four major lineages. Second, chloroplast (psbA) sequence data served to consolidate these PSHs into 13 secondary species hypotheses (SSHs) that showed biologically plausible ranges. Using several lines of evidence (e.g. morphological characters, known species distributions, sequences from type and topotype material), six SSHs were assigned to available species names that included the geographically widespread Phymatolithon calcareum, Lithothamnion corallioides, and L. glaciale; possible identities of other SSHs are discussed. Concordance between SSHs and morphospecies was minimal, highlighting the convenience of DNA barcoding for an accurate identification of maerl specimens. Our survey indicated that a majority of maerl forming species have small distribution ranges and revealed a gradual replacement of species with latitude.     

Species Delimitation and Analysis of Cryptic Species Diversity in the XXI Century

The potentials and limitations of different approaches to revealing species boundaries and describing cryptic species are discussed. Both the traditional methods of species delimitation, mostly based on morphological analysis, and the approaches using molecular markers are considered. Besides, the prospects of species identification using digital image recognition and machine learning are briefly considered. It is concluded that molecular markers provide very important material for species delimitation; the value of these data increases manifold if they can be compared with information on morphology, geographic distribution, and ecological preferences of the studied taxa. In many cases, only a practicing taxonomist who knows his or her group thoroughly can correctly interpret the molecular data and incorporate them into the existing knowledge system in order to make a taxonomic decision.

     

Exploring Species Level Taxonomy and Species Delimitation Methods in the Facultatively Self-Fertilizing Land Snail Genus Rumina (Gastropoda: Pulmonata)

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.     

Differences in Incubation Length and Hatchling Morphology among Five Species of Oviparous Phrynocephalus Lizards （Agamidae） from China

We incubated eggs of five Phrynocephalus species （P. albolineatus, P. axillaries, P. grumgrzimailoi, P. helioscopus and P. przewalskii） at three constant temperatures （24℃, 28℃ and 32℃） to examine differences in incubation length and hatchling morphology among species and among temperature treatments. We combined data from this study with those reported previously for P. frontalis and P. versicolor to examine whether embryonic stage at laying is a causal factor for interspecific variation in incubation length, and whether the phylogenetic relationship inferred from hatchling morphology is consistent with the relationship based on mitochondrial DNA data. Mean values for incubation length differed among the five species studied herein and, in all these five species, incubation length decreased at a decreasing rate as temperature increased. In none of the five species did hatchling size （snout-vent length and body mass） and other morphological variables differ among the three temperature treatments. The seven oviparous Phrynocephalus lizards found in China differ from each other in hatchling morphology, and embryonic stage at laying is a causal factor of inter- and intra-specific variation in incubation length. The phylogenetic relationship inferred from hatchling morphology is not always consistent with the currently known relationship based on mitochondrial DNA data. Data from this study and those reported previously allow the conclusion that any Phrynocephalus species may have its unique position along the axis defined by hatchling morphology.     

苯丙酮尿症常用筛查方法的比较

苯丙酮尿症是一种由于体内缺乏苯丙氨酸羟化酶所引起的遗传性代谢异常疾病,如果能够做到早诊断、早发现、早预防和早治疗,就可以避免患儿以后出现智能发育障碍,因此开展新生儿的筛查工作十分重要。简要介绍了苯丙酮尿症的基本情况以及目前应用于临床的常用筛查方法,如细菌抑制法、荧光法等,并从筛查原理、主要优缺点等方面进行了简单的比较。     

Patient-Specific Metrics of Invasiveness Reveal Significant Prognostic Benefit of Resection in a Predictable Subset of Gliomas

Object

Malignant gliomas are incurable, primary brain neoplasms noted for their potential to extensively invade brain parenchyma. Current methods of clinical imaging do not elucidate the full extent of brain invasion, making it difficult to predict which, if any, patients are likely to benefit from gross total resection. Our goal was to apply a mathematical modeling approach to estimate the overall tumor invasiveness on a patient-by-patient basis and determine whether gross total resection would improve survival in patients with relatively less invasive gliomas.

Methods

In 243 patients presenting with contrast-enhancing gliomas, estimates of the relative invasiveness of each patient''s tumor, in terms of the ratio of net proliferation rate of the glioma cells to their net dispersal rate, were derived by applying a patient-specific mathematical model to routine pretreatment MR imaging. The effect of varying degrees of extent of resection on overall survival was assessed for cohorts of patients grouped by tumor invasiveness.

Results

We demonstrate that patients with more diffuse tumors showed no survival benefit (P = 0.532) from gross total resection over subtotal/biopsy, while those with nodular (less diffuse) tumors showed a significant benefit (P = 0.00142) with a striking median survival benefit of over eight months compared to sub-totally resected tumors in the same cohort (an 80% improvement in survival time for GTR only seen for nodular tumors).

Conclusions

These results suggest that our patient-specific, model-based estimates of tumor invasiveness have clinical utility in surgical decision making. Quantification of relative invasiveness assessed from routinely obtained pre-operative imaging provides a practical predictor of the benefit of gross total resection.     

New Methods for Extraction and Quantitation of Zeins Reveal a High Content of gamma-Zein in Modified opaque-2 Maize

We have developed methods for quantitative extraction and analysis of zeins from maize (Zea mays L.) flour. Extraction involved solubilization of total endosperm proteins in an alkaline buffer containing SDS and 2-mercaptoethanol with subsequent precipitation of nonzein proteins by the addition of ethanol to 70%. Analysis of these proteins by SDS-PAGE with Coomassie blue staining and by Western blotting and ELISA assay with zein antibodies revealed that this extraction method is more quantitative than the traditional Landry-Moureaux procedure, especially for the β- and γ-zeins. This method was used to extract and analyze the zein content of several `Quality Protein Maize' (QPM) varieties developed by the International Maize and Wheat Improvement Center. QPM varieties contain `modifier genes' that confer a vitreous phenotype on opaque-2 genotypes, while maintaining the elevated levels of lysine and tryptophan characteristic of this mutant. This analysis revealed that the QPM types contain 2 to 4 times the amount of the γ-zein than unmodified opaque-2 or normal maize varieties. Possible relationships between the high expression of the γ-zein and the modified opaque phenotype are discussed.     

DNA Sequence Comparison among Closely Related Drosophila Species in the MULLERI Complex

DNA hybridization was used to establish DNA sequence relationships among seven Drosophila species. Single-copy DNA was isolated from four species within the Drosophila mulleri complex, D. mojavensis, D. arizonensis, D. ritae and D. starmeri. These single-copy DNAs were used as tracers to be hybridized with each other and one additional member of the mulleri complex, D. aldrichi, a member of a closely related complex, D. hydei, and a distantly related species, D. melanogaster. Two methods have been used to determine the relatedness between these species: (1) the extent of duplex formed as measured by binding to hydroxyapatite and (2) the thermal stability of the duplexed DNA. Moderately repetitive DNA was purified from these species and used similarly to determine the divergence of this family of sequences. The rate of nucleotide substitution was estimated to be 0.2 +/-, 0.1% base pair change per million years for both single-copy and middle-repetitive DNAs. The size of the D. arizonensis genome, a representative of the mulleri complex, was calculated to be 2.2 X 10(8) base pairs from its kinetic complexity similar to that of D. hydei. The relative amounts (18%) and average reiteration frequency (100 copies) of the middle-repetitive DNA are similar for all Drosophila species studied. Finally, the data are presented in a phylogenetic tree.     

Novel Methods for Analysing Bacterial Tracks Reveal Persistence in Rhodobacter sphaeroides

Tracking bacteria using video microscopy is a powerful experimental approach to probe their motile behaviour. The trajectories obtained contain much information relating to the complex patterns of bacterial motility. However, methods for the quantitative analysis of such data are limited. Most swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. It is therefore necessary to segment observed tracks into swimming and reorientation phases to extract useful statistics. We present novel robust analysis tools to discern these two phases in tracks. Our methods comprise a simple and effective protocol for removing spurious tracks from tracking datasets, followed by analysis based on a two-state hidden Markov model, taking advantage of the availability of mutant strains that exhibit swimming-only or reorientating-only motion to generate an empirical prior distribution. Using simulated tracks with varying levels of added noise, we validate our methods and compare them with an existing heuristic method. To our knowledge this is the first example of a systematic assessment of analysis methods in this field. The new methods are substantially more robust to noise and introduce less systematic bias than the heuristic method. We apply our methods to tracks obtained from the bacterial species Rhodobacter sphaeroides and Escherichia coli. Our results demonstrate that R. sphaeroides exhibits persistence over the course of a tumbling event, which is a novel result with important implications in the study of this and similar species.     

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae,Pucciniales) on Willows in China

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.     

Genetic and Genomic Diversity Studies of Acacia Symbionts in Senegal Reveal New Species of Mesorhizobium with a Putative Geographical Pattern

Acacia senegal (L) Willd. and Acacia seyal Del. are highly nitrogen-fixing and moderately salt tolerant species. In this study we focused on the genetic and genomic diversity of Acacia mesorhizobia symbionts from diverse origins in Senegal and investigated possible correlations between the genetic diversity of the strains, their soil of origin, and their tolerance to salinity. We first performed a multi-locus sequence analysis on five markers gene fragments on a collection of 47 mesorhizobia strains of A. senegal and A. seyal from 8 localities. Most of the strains (60%) clustered with the M. plurifarium type strain ORS 1032^T, while the others form four new clades (MSP1 to MSP4). We sequenced and assembled seven draft genomes: four in the M. plurifarium clade (ORS3356, ORS3365, STM8773 and ORS1032^T), one in MSP1 (STM8789), MSP2 (ORS3359) and MSP3 (ORS3324). The average nucleotide identities between these genomes together with the MLSA analysis reveal three new species of Mesorhizobium. A great variability of salt tolerance was found among the strains with a lack of correlation between the genetic diversity of mesorhizobia, their salt tolerance and the soils samples characteristics. A putative geographical pattern of A. senegal symbionts between the dryland north part and the center of Senegal was found, reflecting adaptations to specific local conditions such as the water regime. However, the presence of salt does not seem to be an important structuring factor of Mesorhizobium species.     

三种阔叶树雌雄异株木材结构的比较

在光学显微镜下较详细地比较了重阳木、大叶刺篱木和红毛丹,雌雄异株木材的结构。结果表明：同一种树木两性间木材结构总体上是很相似的,但有的定量特征差异显著甚至极显著。３个种的树木中,雌株木纤维壁腔比值较雄株的大,而分膈纤维和纤维管胞数量较小。雌株木材结构特点可能表明其木材具较强的机械支持能力,利用于果实的支持 。     

Comparison of Four Species Sensitivity Distribution Methods to Calculate Predicted No Effect Concentrations for Bisphenol A

Bisphenol A (BPA, CAS RN 80-05-7) is a high production volume chemical used as an intermediate in the production of polycarbonate plastic and epoxy resins. During its manufacture and use, some emissions to surface waters are anticipated. Chronic predicted no effect concentrations (PNECs) for aquatic systems are used to support the assessment of potential risks to aquatic organisms in receiving waters. PNECs for a compound are considered protective of populations, communities, and ecosystems. Traditionally, PNECs are derived by taking the lowest no-observed effect concentration (NOEC) from a set of toxicity studies and dividing by an assessment factor (e.g., 10 to 1000). This traditional approach is appropriate for substances with few data, but may not be necessary for substances with many valid studies. For well-studied substances, statistical approaches (i.e., development of Species Sensitivity Distribution or SSD methods) can be used to calculate a PNEC that makes use of the full distribution of available NOEC values. Bisphenol A has an extensive set of aquatic toxicity studies covering diverse taxa including algae, hydra, rotifers, mollusks, crustaceans (both benthic and pelagic), insects, annelids, fish, and amphibians. The full chronic data set was used to calculate PNEC values using four SSD methods: (1) the Hazard Concentration (HC5) approach developed by The Netherlands National Institute of Public Health and the Environment (RIVM), (2) the U.S. Environmental Protection Agency's water quality criteria procedure, (3) SigmaPlot (Systat 2000) commercial software that calculates percentile values, and (4) a distributional method consistent with that used by Environment Canada. Using these approaches, PNEC values for BPA range from 11 to 71 μ g/L. Literature studies suggest that application of an additional assessment factor is unwarranted if an SSD-based PNEC is based on chronic data. SSD-derived PNEC values and the traditionally derived PNEC value of 1.6 μ g/L are then compared to concentrations of BPA that have been measured in North American and European surface waters. Adverse risks to aquatic organisms are not anticipated from measured concentrations of BPA in North American and European surface waters.     

Syngonanthus androgynus,a Striking New Species from South America,its Phylogenetic Placement and Implications for Evolution of Bisexuality in Eriocaulaceae

In the present study, we describe and illustrate a remarkable new species of Syngonanthus from South America (Bolivia, Brazil and Peru). This new species is quickly distinguished from all species in the genus by trimerous and bisexual flowers, a unique set of characteristics in Syngonanthus. Complementary to this study, sequences of 33 species were downloaded from GenBank and four species had sequences newly generated for this study. Molecular phylogenetic analyses based on nuclear ribosomal ITS and the plastid regions psbA-trnH and trnL-F were performed to determine its systematic position. The results have shown S. androgynus closely related to a well-supported clade that has been treated as Syngonanthus sect. Carphocephalus. Floral traits associated with this new plant also were surveyed. Character reconstruction suggests that the bisexual flowers originated independently more than once in the genus. However, trimerous flowers appear to be an ancestral condition of the whole genus.     

不同生境下十七种藓类植物叶的比较解剖学

使用石蜡切片法,对不同生境下的17种藓类植物的叶片进行了解剖观察和比较分析,结果表明不同种类的藓类植物在中肋导水主细胞的有无、厚壁细胞是否分化、中肋细胞层数及细胞密度、叶片细胞层数、叶表附属物、叶片细胞密度等方面存在显著差异。藓类植物叶的解剖结构具有生态适应意义,旱生环境下的藓类植物,叶片细胞胞壁具不同程度的增厚,有些藓类植物叶片具附属结构,藓类植物中肋的有无,反映了对水分吸收和运输方式的不同。例如,荫湿生环境下的羽枝青藓Brachythecium plumosum,其中肋细胞胞壁较薄,无导水主细胞和副细胞的分化,也没有厚壁细胞分化,能够在阴湿环境下吸收水分和养分;钝叶匍灯藓Plagiom niumrostratum具有与旱生藓类植物相似的中肋结构,叶片较厚,中肋具导水主细胞,中肋背面具厚壁细胞,这些特点使该种藓类植物能够分布于间隙性干旱胁迫的环境中;水灰藓Hygro-hypnum luridum叶片纤细柔弱,仅1层细胞,细胞胞壁薄,叶表无附属结构,中肋细胞层数少,无导水主细胞分化,也没有厚壁细胞,这些特点使得水灰藓能够生长在水生环境中;东亚小金发藓Pogonatum inflexum和波叶仙鹤藓Atrich umundulatum的叶腹面覆盖着栉片,东亚砂藓Racomitrium japonicum、大羽藓Thuidium cym-bifolium、福氏蓑藓Macromitrium ferriei、东亚短颈藓Diphyscium fulvifolium、扭口藓Barbula unguiculata和角齿藓Ceratodon purpureus的叶片表面有乳头状突起或疣状物,这些附属结构使它们能够适应于旱生的环境中。     

Comparison of Two New Robust Parameter Estimation Methods for the Power Function Distribution

Estimation of any probability distribution parameters is vital because imprecise and biased estimates can be misleading. In this study, we investigate a flexible power function distribution and introduced new two methods such as, probability weighted moments, and generalized probability weighted methods for its parameters. We compare their results with L-moments, trimmed L-moments by a simulation study and a real data example based on performance measures such as, mean square error and total deviation. We concluded that all the methods perform well in the case of large sample size (n>30), however, the generalized probability weighted moment method performs better for small sample size.     

Detection of Hippurate Hydrolase among Bacillus Species by Thin Layer Chromatography and other Methods

S ummary . The ability of 111 isolates of 33 species of Bacillus to hydrolyze hippurate into benzoic acid and glycine was tested. All, other than the strains classified as B. badius, B. cereus var. mycoides, B. cereus var. thuringiensis, B. macquariensis, B. medusa, B. pacificus, B. psychrosaccharolyticus and some B. macerans and B. polymyxa strains, were positive by thin layer chromatography (TLC). Of the 4 methods compared (hippurate agar, the sulphuric acid method, precipitation with ferric chloride and TLC), only the TLC procedure allowed a rapid and definite detection of the hydrolytic product, benzoic acid. The use of hippurate hydrolase as an additional diagnostic aid in the differentiation of aerobic spore-forming bacilli is discussed. Other characters were used to compare some recently described Bacillus spp. The combination B. cereus subsp. medusa n. subsp. is proposed.     

Comparison of Methods for Tuberculosis Bacteriology

To improve efficiency of isolation of tubercle bacilli from clinical specimens, the following recommendations are presented. (i) Employ multiple specimens consisting of a combination of morning sputums for the early detection of positives, along with 24-hr sputum pools for the greatest total yield of positives. (ii) When timing is rigorously controlled, Zephiran-trisodium phosphate and sodium hydroxide-acetylcysteine are comparable, but if timing cannot rigidly be controlled, employ the Zephiran-trisodium phosphate digestion procedure to allow the greatest freedom in exposure time with the lowest kill rate to tubercle bacilli. (iii) Employ both an agar medium incubated in 5% CO₂, for the early detection of positives as well as positives in the presence of contaminants, and an egg medium, preferably with CO₂, to increase the yield of positives.