Species identification of Alnus (Betulaceae) using nrDNA and cpDNA genetic markers

One nuclear and three chloroplast DNA regions (ITS, rbcL, matK and trnH-psbA) were used to identify the species of Alnus (Betulaceae). The results showed that 23 out of all 26 Alnus species in the world, represented by 131 samples, had their own specific molecular character states, especially for three morphologically confused species (Alnus formosana, Alnus japonica and Alnus maritima). The discriminating power of the four markers at the species level was 10% (rbcL), 31.25% (matK), 63.6% (trnH-psbA) and 76.9% (ITS). For ITS, the mean value of genetic distance between species was more than 10 times the intraspecific distance (0.009%), and 13 species had unique character states that differentiated them from other species of Alnus. The trnH-psbA region had higher mean values of genetic distance between and within species (2.1% and 0.68% respectively) than any other region tested. Using the trnH-psbA region, 13 species are distinguished from 22 species, and seven species have a single diagnostic site. The combination of two regions, ITS and trnH-psbA, is the best choice for DNA identification of Alnus species, as an improvement and supplement for morphologically based taxonomy. This study illustrates the potential for certain DNA regions to be used as novel internet biological information carrier through combining DNA sequences with existing morphological character and suggests a relatively reliable and open taxonomic system based on the linked DNA and morphological data.     

Ontogeny and phylogeny in temnospondyls: a new method of analysis

A new method has been devised to compare the ontogeny and phylogeny of some of the better documented tetrapods from the Stephanian to the Trias. This approach is based on global parsimony analysis of several temnospondyl amphibians, in which some ontogenetic sequences have been highlighted. Forty-one homologous morphoanatomical character states have been separated into larval, juvenile and adult stages of each of six tetrapod species. The taxonomic congruence (TC) approach involves comparing trees based on larval, juvenile and adult character states. These so-called 'ontotrees' are not congruent, either in their topologies or in the distribution of the character states. The total evidence (TE) approach involves a combined analysis of all the character states observed in the various growth stages of the taxa, and is secondarily used in order to test this taxonomic incongruence. In this case, the TE result corroborates the TC analysis: the TE tree is robust and reveals a few homoplasies which cause the taxonomic incongruence. This is interpreted as either the result of heterochronic events in temnospondyl evolution, or as the product of inaccurate identification of larval and metamorphic fossil forms.     

Oligonucleotide fingerprint identification for microarray-based pathogen diagnostic assays

MOTIVATION: Advances in DNA microarray technology and computational methods have unlocked new opportunities to identify 'DNA fingerprints', i.e. oligonucleotide sequences that uniquely identify a specific genome. We present an integrated approach for the computational identification of DNA fingerprints for design of microarray-based pathogen diagnostic assays. We provide a quantifiable definition of a DNA fingerprint stated both from a computational as well as an experimental point of view, and the analytical proof that all in silico fingerprints satisfying the stated definition are found using our approach. RESULTS: The presented computational approach is implemented in an integrated high-performance computing (HPC) software tool for oligonucleotide fingerprint identification termed TOFI. We employed TOFI to identify in silico DNA fingerprints for several bacteria and plasmid sequences, which were then experimentally evaluated as potential probes for microarray-based diagnostic assays. Results and analysis of approximately 150 in silico DNA fingerprints for Yersinia pestis and 250 fingerprints for Francisella tularensis are presented. AVAILABILITY: The implemented algorithm is available upon request.     

DNA barcoding reveals Mexican diversity within the freshwater leech genus Helobdella (Annelida: Glossiphoniidae)

We investigated the genetic distances and taxonomic status among species of Helobdella, a genus of non-blood-feeding leeches, based on mitochondrial cytochrome c oxidase subunit I sequences. Sampling included 20 specimens representing nine nominal species collected in 11 states in Mexico as well as previously published sequences of different species of Helobdella from several places. A neighbor-joining tree, as well as identification of diagnostic nucleotides, was used to suggest the presence of seven species of Helobdella in Mexico including potentially two undescribed forms.     

Molecular and morphometric evidence for separate species of Uncinaria (Nematoda: Ancylostomatidae) in California sea lions and northern fur seals: hypothesis testing supplants verification

California sea lions (Zalophus californianus) and northern fur seals (Callorhinus ursinus) are each believed to host distinct hookworm species (Uncinaria spp.). However, a recent morphometric analysis suggested that a single species parasitizes multiple pinniped hosts, and that the observed differences are host-induced. To explore the systematics of these hookworms and test these competing hypotheses, we obtained nucleotide sequences of nuclear ribosomal DNA (D2/D3 28S, D18/D19 28S, and internal transcribed spacer [ITS] regions) from 20 individual hookworms parasitizing California sea lion and northern fur seal pups where their breeding grounds are sympatric. Five individuals from an allopatric population of California sea lions were also sampled for ITS-1 and D18/D19 28S sequences. The 28S D2/D3 sequences showed no diagnostic differences among hookworms sampled from individual sea lions and fur seals, whereas the 28S D18/D19 sequences had one derived (apomorphic) character demarcating hookworms from northern fur seals. ITS sequences were variable for 7 characters, with 4 derived (apomorphic) states in ITS-1 demarcating hookworms from California sea lions. Multivariate analysis of morphometric data also revealed significant differences between nematodes representing these 2 host-associated lineages. These results indicate that these hookworms represent 2 species that are not distributed indiscriminately between these host species, but instead exhibit host fidelity, evolving independently with each respective host species. This evolutionary approach to analyzing sequence data for species delimitation is contrasted with similarity-based methods that have been applied to numerous diagnostic studies of nematode parasites.     

ON COMBINING PROTEIN SEQUENCES AND NUCLEIC ACID SEQUENCES IN PHYLOGENETIC ANALYSIS: THE HOMEOBOX PROTEIN CASE

Abstract— Amino acid encoding genes contain character state information that may be useful for phylogenetic analysis on at least two levels. The nucleotide sequence and the translated amino acid sequences have both been employed separately as character states for cladistic studies of various taxa, including studies of the genealogy of genes in multigene families. In essence, amino acid sequences and nucleic acid sequences are two different ways of character coding the information in a gene. Silent positions in the nucleotide sequence (first or third positions in codons that can accrue change without changing the identity of the amino acid that the triplet codes for) may accrue change relatively rapidly and become saturated, losing the pattern of historical divergence. On the other hand, non-silent nucleotide alterations and their accompanying amino acid changes may evolve too slowly to reveal relationships among closely related taxa. In general, the dynamics of sequence change in silent and non-silent positions in protein coding genes result in homoplasy and lack of resolution, respectively. We suggest that the combination of nucleic acid and the translated amino acid coded character states into the same data matrix for phylogenetic analysis addresses some of the problems caused by the rapid change of silent nucleotide positions and overall slow rate of change of non-silent nucleotide positions and slowly changing amino acid positions. One major theoretical problem with this approach is the apparent non-independence of the two sources of characters. However, there are at least three possible outcomes when comparing protein coding nucleic acid sequences with their translated amino acids in a phylogenetic context on a codon by codon basis. First, the two character sets for a codon may be entirely congruent with respect to the information they convey about the relationships of a certain set of taxa. Second, one character set may display no information concerning a phylogenetic hypothesis while the other character set may impart information to a hypothesis. These two possibilities are cases of non-independence, however, we argue that congruence in such cases can be thought of as increasing the weight of the particular phylogenetic hypothesis that is supported by those characters. In the third case, the two sources of character information for a particular codon may be entirely incongruent with respect to phylogenetic hypotheses concerning the taxa examined. In this last case the two character sets are independent in that information from neither can predict the character states of the other. Examples of these possibilities are discussed and the general applicability of combining these two sources of information for protein coding genes is presented using sequences from the homeobox region of 46 homeobox genes fromDrosophila melanogasterto develop a hypothesis of genealogical relationship of these genes in this large multigene family.     

Rooting the tree of life using nonubiquitous genes

Insertion and deletion (indel)-based analyses have great potential for rooting the tree of life, but their use has been limited because they require ubiquitous sequences that have not been horizontally/laterally transferred. Very few such sequences exist. Here we describe and demonstrate a new algorithm that can use nonubiquitous sequences for rooting. This algorithm, top-down indel rooting, uses the traditional logical framework of indel rooting, but by considering gene gains and losses in addition to indel gains and losses, it is able to analyze incomplete data sets. The method is demonstrated using theoretical examples and incomplete gene sets. In particular, it is applied to the well-studied Hsp70/MreB indel, a sequence set thought to have been compromised by gene transfers from Firmicutes to archaebacteria. By sequentially assigning all observable character states, including gene absences, to the questionable archaebacterial Hsp70 and MreB sequences, we demonstrate that this gene set robustly excludes the root of the tree of life from the Gram-negative, double-membrane prokaryotes independently of the archaeal character states. There are very few ubiquitous paralog gene sets, and most of them contain compromised data. The ability of top-down rooting to use incomplete and/or compromised gene sets promises to make rooting analyses more robust and to greatly increase the number of useful indel sets.     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.     

Comprehensive DNA signature discovery and validation

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.     

On gaps.

Gaps result from the alignment of sequences of unequal length during primary homology assessment. Viewed as character states originating from particular biological events (mutation), gaps contain historical information suitable for phylogenetic analysis. The effect of gaps as a source of phylogenetic data is explored via sensitivity analysis and character congruence among different data partitions. Example data sets are provided to show that gaps contain important phylogenetic information not recovered by those methods that omit gaps in their calculations. However, gap cost schemes are arbitrary (although they must be explicit) and thus data exploration is a necessity of molecular analyses, while character congruence is necessary as an external criterion for hypothesis decision.     

Design of character‐based DNA barcode motif for species identification: A computational approach and its validation in fishes

The DNA barcodes are generally interpreted using distance‐based and character‐based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance‐based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character‐based approach more accurately defines this using a unique set of nucleotide characters. The character‐based analysis of full‐length barcode has some inherent limitations, like sequencing of the full‐length barcode, use of a sparse‐data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154‐bp fragment, from the transversion‐rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species‐specific barcode motifs for 109 species by the character‐based method, which successfully identifies the correct species using a pattern‐matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species‐specific mini‐barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini‐barcode approach will greatly benefit the field‐based system of rapid species identification.     

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.     

Examining morphological and molecular diagnostic character states of Cichorium intybus L. (Asteraceae) and C. spinosum L.

The inter- and intraspecific variability of Cichorium intybus L. was examined to evaluate potential morphological and molecular diagnostic character states. Two diagnostic and one overlapping morphological character clearly delimit the two species C. intybus and C. spinosum. All applied molecular methods (ITS, AFLP, Microsatellites) failed to significantly discriminate between these accepted species. As the morphological traits are fixed and heritable, criteria for species delimitation are fulfilled. These traits, however, are apparently due to mutations of a few crucial loci affecting the morphological diagnostic character states. Intraspecific variability within C. intybus revealed to be highly influenced by plastic response to local environmental factors and subspecific delimitation cannot be supported.     

Mitochondrial genome sequence of <Emphasis Type="Italic">Unionicola parkeri</Emphasis> (Acari: Trombidiformes: Unionicolidae): molecular synapomorphies between closely-related <Emphasis Type="Italic">Unionicola</Emphasis> gill mites

The mitochondrial genome of Unionicola parkeri is a 14,734 bp circular DNA molecule. The sequence and annotation revealed a unique gene order, related to but distinct from the gene order in the closely related species U. foili. Mitochondrial tRNA sequences annotated in this genome predict non-canonical secondary structures for these molecules. The continuing patterns of unique gene orders and unusual tRNA structures in the Trombidiformes in general and Unionicola in particular support the use of phylogenetic approaches that use these types of molecular features as shared, derived character states. Further progress in using these molecular character states to reconstruct phylogeny will depend on careful annotation, especially of tRNA genes.     

A System for Heart Sounds Classification

The future of quick and efficient disease diagnosis lays in the development of reliable non-invasive methods. As for the cardiac diseases – one of the major causes of death around the globe – a concept of an electronic stethoscope equipped with an automatic heart tone identification system appears to be the best solution. Thanks to the advancement in technology, the quality of phonocardiography signals is no longer an issue. However, appropriate algorithms for auto-diagnosis systems of heart diseases that could be capable of distinguishing most of known pathological states have not been yet developed. The main issue is non-stationary character of phonocardiography signals as well as a wide range of distinguishable pathological heart sounds. In this paper a new heart sound classification technique, which might find use in medical diagnostic systems, is presented. It is shown that by combining Linear Predictive Coding coefficients, used for future extraction, with a classifier built upon combining Support Vector Machine and Modified Cuckoo Search algorithm, an improvement in performance of the diagnostic system, in terms of accuracy, complexity and range of distinguishable heart sounds, can be made. The developed system achieved accuracy above 93% for all considered cases including simultaneous identification of twelve different heart sound classes. The respective system is compared with four different major classification methods, proving its reliability.