Allopatry as a Gordian Knot for Taxonomists: Patterns of DNA Barcode Divergence in Arctic-Alpine Lepidoptera

Many cold adapted species occur in both montane settings and in the subarctic. Their disjunct distributions create taxonomic complexity because there is no standardized method to establish whether their allopatric populations represent single or different species. This study employs DNA barcoding to gain new perspectives on the levels and patterns of sequence divergence among populations of 122 arctic-alpine species of Lepidoptera from the Alps, Fennoscandia and North America. It reveals intraspecific variability in the barcode region ranging from 0.00–10.08%. Eleven supposedly different species pairs or groups show close genetic similarity, suggesting possible synonymy in many cases. However, a total of 33 species show evidence of cryptic diversity as evidenced by the presence of lineages with over 2% maximum barcode divergence in Europe, in North America or between the two continents. Our study also reveals cases where taxonomic names have been used inconsistently between regions and exposes misidentifications. Overall, DNA barcodes have great potential to both increase taxonomic resolution and to make decisions concerning the taxonomic status of allopatric populations more objective.     

Geographic structure of chloroplast DNA polymorphisms in European oaks

Chloroplast DNA polymorphisms have been detected by the conventional Southern-blotting hybridization method in four species of European oaks (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica). Three polymorphisms, shared by at least three of these species, can be scored directly in ethidium bromidestained gels and were used in a broad survey of the level of differentiation of the oak species and of their pattern of genetic structure in western Europe. The highly significant geographic variation and the high genetic differentiation (Gst=0.895, S_Gst=0.025) indicate a low level of cytoplasmic gene flow. We conclude that cytoplasmic genomes are well suited for the reconstruction of past migrational routes of such a complex of species.     

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5ʹ (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.     

栗属中国特有种居群的遗传多样性及地域差异

Genetic variability in 30 populations of three endemic Castanea (Tourn.) L. species (C. moUissima BI., C. seguinii Dode. and C. henryi (Skan) Rehd. et Wils. ) in China was investigated using isoelectric focusing in thin-layer polyacrylamide slab gels at 20 loci coding for 12 enzyme systems. C. mollissima was found to possess a significant higher value of genetic variability than that of the other two species. The percentage of polymorphic loci (P) and the expected heterozygosity (He) were 90.0% and 0. 311 at the species level, respectively; while P = 84.7 %, He = 0.295 at the population level. Comparison of the population genetic variability of C. mollissima in three regions of China, the populations from the Yangtze River valley showed a markedly higher mean expected gene heterozygosity. The results provided additional evidence that the Yangtze River valley, particularly in Shennongjia area, was the center of the genetic diversity of C. moUissima. Genetic relationships among populations and species were assessed by Nei genetic identity (I) and standard genetic distance (D), suggesting that C. mollissima and C. seguinii have a closer relationship, and genetic distances were correlated with geographical distances among populations. Genetic differentiation between populations in C. mollissima, C. seguinii and C. henryi was 7.5%, 10.9% and 22.1%, respectively, and the gene flow rate (Nm) was 3.20, 2.05 and 0.88 respectively. The study provides useful imfonnation for understanding of the origin and evolutionary events in genus Castanea and planning an effective conservation strategy.     

Genetic variation of mitochondrial DNA in Chinese populations of Pectinophora gossypiella (Lepidoptera: Gelechiidae)

The pink bollworm Pectinophora gossypiella is an invasive pest insect that has successfully established populations in many cotton growing regions around the world. In this study, the genetic diversity and population structure of Chinese populations of P. gossypiella were evaluated using mitochondrial DNA sequence data (COII and Nad4). For comparison, individuals of Pakistan and America were also sequenced at the same two mtDNA regions. Extremely low genetic variation was observed in the two mitochondrial regions among all populations examined. Most of the populations harbored only one to two haplotypes. Although the Nad4 region showed relatively high haplotype diversity and nucleotide variation, ranging from 0.363 to 0.591 and from 0.00078 to 0.00140, respectively, there were only three haplotypes observed in this region. COII and Nad4 haplotype networks shaved one or two common haplotype(s) forming the center of a star-shaped phylogeny. Pairwise tests showed that most of the populations were not significantly differentiated from each other. The Chinese populations were differentiated from the Pakistani and American populations in the Nad4 region. The low level of population genetic variation of P. gossypiella is attributed to invasion bottlenecks, which may have been subsequently strengthened by its nonmigratory biology and the mosaic pattern of agricultural activities.     

Optimal Testing for Serial Correlation in a Large Number of Small Samples

In recent years, there has been an increased awareness of the potential one-sided nature of many testing problems in applied sciences. Usually, these testing problems can be reduced, either by conditioning on sufficient statistics or by invariant techniques. COX and SOLOMON (1988) considered testing the serial correlation coefficient of a stationary first order autoregressive process and concentrated on four independent samples, with each of size three. We outline a general method for testing the serial correlation coefficient, using locally best invariant, point optimal invariant and locally most mean powerful invariant test procedures. The first procedure optimizes power near the null hypothesis, the second optimizes it at a pre-determined point away from the null while the third optimizes the average curvature of the power hypersurface in the neighbourhood of the null hypothesis.     

Role of DNA Sequences in Genetic Recombination in the Iso-1-Cytochrome c Gene of Yeast. I. Discrepancies between Physical Distances and Genetic Distances Determined by Five Mapping Procedures

Recombination rates have been examined in two-point crosses of various defined cyc1 mutants using five mapping methods. Nucleotide sequences of mutant codons were identified in previous studies from alterations in functional iso-1-cytochromes c produced by intragenic revertants. Heteroallelic diploids were analyzed for rates of mitotic recombination that occurred spontaneously and that were induced with x-rays, ultraviolet light and the near-ultraviolet light emitted by sunlamps, as well as rates of meiotic recombination that occur after sporulation. Frequencies of both mitotic and meiotic recombination do not necessarily correspond with physical distances separating altered nucleotides. The most extreme discrepancy involved two adjacent intervals of thirteen basepairs which differed approximately thirty-fold in their spontaneous and X-ray-induced recombination rates. Marked disproportions between genetic and physical distances appear to be due to the interaction of the two nucleotide sequences in the heteroallelic combination and not to the sequences of the mutant codons alone. Recombination values that were obtained by all five methods could not be used to establish to correct order of mutant sitesmrelationships of the recombination rates for the various pairwise crosses are different after mitosis from those after meiosis, suggesting that these two recombinational processes are to some extent different in their dependence on particular nucleotide configurations. On the other hand, the relationships of the rates induced by UV-, sunlamp- and X-irradiation were identical or very similar. In addition to the intrinsic properties of the alleles affecting frequencies of mitotic and meiotic recombination rates, two- to threefold variations in recombination rates could be attributed to genetic backgrounds.     

Daily Online Testing in Large Classes: Boosting College Performance while Reducing Achievement Gaps

An in-class computer-based system, that included daily online testing, was introduced to two large university classes. We examined subsequent improvements in academic performance and reductions in the achievement gaps between lower- and upper-middle class students in academic performance. Students (N = 901) brought laptop computers to classes and took daily quizzes that provided immediate and personalized feedback. Student performance was compared with the same data for traditional classes taught previously by the same instructors (N = 935). Exam performance was approximately half a letter grade above previous semesters, based on comparisons of identical questions asked from earlier years. Students in the experimental classes performed better in other classes, both in the semester they took the course and in subsequent semester classes. The new system resulted in a 50% reduction in the achievement gap as measured by grades among students of different social classes. These findings suggest that frequent consequential quizzing should be used routinely in large lecture courses to improve performance in class and in other concurrent and subsequent courses.     

青海省6种裂腹鱼COI基因作为DNA条形码的适用性初探

青海省裂腹鱼鱼类至少有20种,占土著鱼类的40%以上,具有重要的生态价值。由于生态环境的恶化和人为因素的干扰,很多物种群已濒临灭绝。快速和准确的物种鉴定对于这些物种的保护至关重要,而基于形态学的传统分类法很难满足这一需求。因此,本研究通过DNA条形码技术初步探讨了在青海省裂腹鱼物种鉴定中的适用性。本研究中,测序获得了青海26尾裂腹鱼(6个物种)的细胞色素c氧化酶亚基I (COI)基因,并经GenBank数据库进行比对;基于K2P模型分析COI序列变异;运用贝叶斯法(BI)和最大似然法(ML)构建系统发育树。结果显示,6种裂腹鱼COI序列检测得到15个单倍型,且各物种间无共享的单倍型。基于K2P模型,最大种内遗传距离和最小种间遗传距离分别为(0.621±0.297)%和(2.792±0.644)%。种间平均遗传距离为(12.205±1.307)%,约为种内平均遗传距离((0.327±0.162)%)的37倍,表明各物种的COI序列间已经形成明显"条形码间隙"。AMOVA分析显示,遗传变异主要来自种间,约占97.05%;FST=0.970 54,p<0.01,说明各物种间分化程度极高。此外,基于BI和ML方法构建的系统树具有一致的拓扑结构,分辨率较高;各物种均单独聚为一个发育枝,拓扑结构合理。以上结果表明,COI基因作为DNA条形码在青海裂腹鱼物种鉴定中具有较高的适用性。     

Genetic variation among European strains of Saccharomyces paradoxus: results from DNA fingerprinting

We used microsatellite fingerprinting and RAPD analysis to characterize 28 wild European strains of Saccharomyces paradoxus. In contrast to our results from a previous allozyme survey [Naumov et al. Int. J. Syst. Bacteriol. 47: 341-344 (1997a)], these methods revealed extensive genetic variation. The RAPD primers 5'AATCGGGCTG and 5'GGGTAACGCC and the microsatellite primer (GTG)5 yielded reproducible amplification patterns of sufficient clarity and variability to distinguish individual strains from the wild. UPGMA analysis tended to group the strains according to climatic and geographic origin. A comparative study of Ty1 sequence having multiple chromosomal location was also done. Each wild S. paradoxus isolate shows a unique hybridization pattern allowing discrimination to the strain level.     

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

Introduction

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity.

Methods

Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood.

Results

The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood.

Conclusion

Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.     

极小种群野生植物海南风吹楠的遗传多样性研究

为探讨海南风吹楠(Horsfieldia hainanensis)的濒危原因,利用限制性酶切位点相关的DNA测序技术(RAD-seq)开发单核苷酸多态性(SNPs),评估居群的遗传多样性和遗传结构.结果表明,海南风吹楠的遗传多样性较低(Ho=0.167),其中BWL居群表现出最高的遗传多样性;居群间存在中等程度的遗传分...     

Genetic Variation and Geographic Differentiation in Mitochondrial DNA of the Horseshoe Crab, LIMULUS POLYPHEMUS

Restriction site variation in mitochondrial DNA (mtDNA) of the horseshoe crab (Limulus polyphemus) was surveyed in populations ranging from New Hampshire to the Gulf Coast of Florida. MtDNA clonal diversity was moderately high, particularly in southern samples, and a major genetic "break" (nucleotide sequence divergence approximately 2%) distinguished all sampled individuals which were north vs. south of a region in northeastern Florida. The area of genotypic divergence in Limulus corresponds to a long-recognized zoogeographic boundary between warm-temperate and tropical marine faunas, and it suggests that selection pressures and/or gene flow barriers associated with water mass differences may also influence the evolution of species widely distributed across such transition zones. On the other hand, a comparison of the mtDNA divergence patterns in Limulus with computer models involving stochastic lineage extinction in species with limited gene flow demonstrates that deterministic explanations need not necessarily be invoked to account for the observations. Experiments to distinguish stochastic from deterministic possibilities are suggested. Overall, the pattern and magnitude of mtDNA differentiation in horseshoe crabs is very similar to that typically reported for freshwater and terrestrial species assayed over a comparable geographic range. Results demonstrate for the first time that, geographically, at least some continuously distributed marine organisms can show considerable mtDNA genetic differentiation.     

Delineating Species with DNA Barcodes: A Case of Taxon Dependent Method Performance in Moths

The accelerating loss of biodiversity has created a need for more effective ways to discover species. Novel algorithmic approaches for analyzing sequence data combined with rapidly expanding DNA barcode libraries provide a potential solution. While several analytical methods are available for the delineation of operational taxonomic units (OTUs), few studies have compared their performance. This study compares the performance of one morphology-based and four DNA-based (BIN, parsimony networks, ABGD, GMYC) methods on two groups of gelechioid moths. It examines 92 species of Finnish Gelechiinae and 103 species of Australian Elachistinae which were delineated by traditional taxonomy. The results reveal a striking difference in performance between the two taxa with all four DNA-based methods. OTU counts in the Elachistinae showed a wider range and a relatively low (ca. 65%) OTU match with reference species while OTU counts were more congruent and performance was higher (ca. 90%) in the Gelechiinae. Performance rose when only monophyletic species were compared, but the taxon-dependence remained. None of the DNA-based methods produced a correct match with non-monophyletic species, but singletons were handled well. A simulated test of morphospecies-grouping performed very poorly in revealing taxon diversity in these small, dull-colored moths. Despite the strong performance of analyses based on DNA barcodes, species delineated using single-locus mtDNA data are best viewed as OTUs that require validation by subsequent integrative taxonomic work.     

携带大片段BIBAC克隆在不同农杆菌中的遗传稳定性研究

大片段克隆在农杆菌中的稳定性是利用可转化大片段载体进行农杆菌介导遗传转化的关键问题.选用插入片段分子质量分别为150kb和50kb的BIBAC克隆,测定了在3种农杆菌AGL-1,EHA105和LBA4404中的遗传稳定性.从第1、3、5次继代培养的农杆菌中抽提的质粒及酶切结果显示,由于农杆菌背景质粒的干扰,难以判断质粒的降解与否.将农杆菌质粒再转化到大肠杆菌宿主中,发现来自农杆菌AGL-1和EHA105的质粒出现了明显的降解,片段变小,而来自农杆菌LBA4404的质粒没有变化.结果表明,大片段BIBAC质粒在不同农杆菌菌株中的稳定性不同,在农杆菌LBA4404中比较稳定,适合用于遗传转化.     

Large Genetic Change at Small Fitness Cost in Large Populations of Drosophila Melanogaster Selected for Wind Tunnel Flight: Rethinking Fitness Surfaces

The fitness effects of extreme genetic change by selection were studied in large populations subjected to prolonged, intense selection. Two replicate populations of Drosophila melanogaster, with estimated effective sizes 500 <= N(e) <= 1000, were selected for increased performance in a wind tunnel, selecting on average the fastest 4.5% of flies. The mean apparent flying speed of both lines increased from ~2 to 170 cm/sec and continued to respond at diminishing rates, without reaching a plateau, for 100 generations. Competitive fitness tests in generations 50 and 85 showed minimal or no fitness loss in selected lines compared to controls. Sublines relaxed in generations 65 and 85 showed minimal or no regression in apparent flying speed. Hybrid lines, from a cross of selected X control lines in generation 75, responded to reselection saltationally, showing that the chromosomes of the selected lines had been assembled from alleles at many loci, from many different chromosomes in the base population. Thus, major genetic change was achieved, but without the costs usually associated with strong directional selection. Large population size has been interpreted, in opposing models, as either a brake or an accelerator in its effects on long-term change by selection. These results favor the second model, and challenge the concept of rugged fitness surfaces underlying the first model.     

DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用

为了探讨DNA条形码技术在夜蛾物种鉴定中的可行性, 本研究利用条形码通用引物扩增了北京百花山地区43种夜蛾75个样本的线粒体细胞色素C氧化酶亚基I （mitochondrial cytochrome c oxidase subunit I, COI）基因序列, 以Kimura双参数模型进行种内种间遗传距离分析、 使用邻接法（neighbor-joining, NJ）和最大简约法（maximum parsimony, MP）分别构建系统发育树, 并利用分子序列差异阈值对样本进行分子可操作分类单元（molecular defined operational taxonomic units, MOTU）划分。结果表明： 所有夜蛾种类通过系统发育树可以成功区分; 种内平均遗传距离(0.03%)远远小于种间平均遗传距离(11.29%); 采用较为保守的1%的序列差异阈值将75个夜蛾样本分为42个MOTU, 正确率为95%, 除了MOTU04包含2个物种外, 剩余41个MOTU与形态种呈现一一对应的关系。结果显示, 基于COI基因的DNA条形码对于本研究中所涉及的夜蛾具有较好的区分, 可以作为一种有效的工具在夜蛾科昆虫物种鉴定中进行应用。     

Geographic variation of sex pheromone and mitochondrial DNA in Diatraea saccharalis (Fab., 1794) (Lepidoptera: Crambidae)

(9Z,11E)-hexadecadienal and (Z11)-hexadecenal, the main sex pheromone components of the sugarcane borer, Diatraea saccharalis, were identified and quantified from four Brazilian and one Colombian populations using GC-EAD, GC-MS and GC analyses. Three different ratios were observed, 9:1, 6:1, and 3:1. The pheromone concentration for the major component, (9Z,11E)-hexadecadienal, varied from 6.8 ng/gland to 21.9 ng/gland and from 1.7 ng/gland to 6.5 to the minor component, (Z11)-hexadecenal. The 25 D. saccharalis cytochrome oxidase II sequences that were analyzed showed low intra-specific variation and represented only 11 haplotypes, with the most frequent being the one represented by specimens from São Paulo, Paraná, and Pernambuco states. Specimens from Colombia showed the highest genetic divergence from the others haplotypes studied. Data on the genetic variability among specimens, more than their geographic proximity, were in agreement with data obtained from analyses of the pheromone extracts. Our data demonstrate a variation in pheromone composition and a covariation in haplotypes of the D. saccharalis populations studied.     

Effects of Hybridization and Evolutionary Constraints on Secondary Metabolites: The Genetic Architecture of Phenylpropanoids in European Populus Species

The mechanisms responsible for the origin, maintenance and evolution of plant secondary metabolite diversity remain largely unknown. Decades of phenotypic studies suggest hybridization as a key player in generating chemical diversity in plants. Knowledge of the genetic architecture and selective constraints of phytochemical traits is key to understanding the effects of hybridization on plant chemical diversity and ecological interactions. Using the European Populus species P. alba (White poplar) and P. tremula (European aspen) and their hybrids as a model, we examined levels of inter- and intraspecific variation, heritabilities, phenotypic correlations, and the genetic architecture of 38 compounds of the phenylpropanoid pathway measured by liquid chromatography and mass spectrometry (UHPLC-MS). We detected 41 quantitative trait loci (QTL) for chlorogenic acids, salicinoids and flavonoids by genetic mapping in natural hybrid crosses. We show that these three branches of the phenylpropanoid pathway exhibit different geographic patterns of variation, heritabilities, and genetic architectures, and that they are affected differently by hybridization and evolutionary constraints. Flavonoid abundances present high species specificity, clear geographic structure, and strong genetic determination, contrary to salicinoids and chlorogenic acids. Salicinoids, which represent important defence compounds in Salicaceae, exhibited pronounced genetic correlations on the QTL map. Our results suggest that interspecific phytochemical differentiation is concentrated in downstream sections of the phenylpropanoid pathway. In particular, our data point to glycosyltransferase enzymes as likely targets of rapid evolution and interspecific differentiation in the ‘model forest tree’ Populus.