Species Identification of Multimammate Rats of the Genus <Emphasis Type="Italic">Mastomys</Emphasis> (Rodentia: Muridae) in Eastern Africa Using PCR Typing of Cytochrome <Emphasis Type="Italic">b</Emphasis> Gene Fragments

The multimammate rats of the genus Mastomys are widespread throughout sub-Saharan Africa. They are the major agricultural pests and reservoirs of many infections dangerous to humans. A simple and accurate species identification of multimammate rats is crucial in ecological and epidemiological studies; however, it is complicated by the absence of pronounced morphological differentiation between Mastomys species. We describe a simple molecular assay based on PCR typing of the cytochrome b gene fragments as a method that allows fast genotyping of a large number of samples without sequencing to distinguish Mastomys species of East Africa.     

Identification of endangered Mediterranean cyprinodontiform fish by means of DNA inter-simple sequence repeats (ISSRs)

We used inter-simple sequence repeat (ISSR) technique to obtain species-specific molecular markers for the cyprinodontiform fish Valencia hispanica, Valencia letourneuxi and Aphanius fasciatus. The aims were (i) to assess the effectiveness of ISSRs in discriminating the three species and (ii) to identify tissues of two unidentified fish suspected to belong to one of the three above species by comparing ISSR genotypes. Nine ISSR primers produced 101 scorable polymorphic loci with a large number of species-specific bands. UPGMA cluster analysis and nonmetric multidimensional scaling of inter-individual genetic dissimilarities were consistent in separating the three species and attributing the unidentified individuals to V. hispanica. This result was corroborated by the assignment test. ISSRs represent an attractive tool for specimen and species identification. We recommend their use, coupled with appropriate statistical analyses, in species identification when only small portions of the organisms are available.     

Species identification using a small nuclear gene fragment: application to sympatric wild carnivores from South-western Europe

Species identification is essential for non-invasive studies of elusive and rare animals, and for detecting illegal harvest or trade of wildlife species. However, most molecular tests identify only a limited number of species or require multiple laboratory steps to distinguish many taxa. Additionally, most protocols use mitochondrial DNA being, therefore, especially prone to problems such as nuclear insert copies, high intraspecific diversity or heteroplasmy. Here, we developed a molecular test based on the polymorphisms detected on a small nuclear gene fragment (221 bp of the IRBP -Interphotoreceptor Retinoid-Binding Protein- exon 1). This fragment revealed 51 variable sites (including 12 non-synonymous and 19 species-specific sites), which enabled the successful distinction of all 16 carnivore species native to South-western Europe. A SSCP (Single-Strand Conformational Polymorphism) gel electrophoresis technique was also optimized to allow the simple and inexpensive application of this molecular test. Sequences and SSCP profiles were consistent in identifying a total of 387 samples, including faeces (172) and hairs (17) collected non-invasively in the field. Due to its low cost, simplicity, and wide range of identifiable species, this test shows great promise to facilitate studies in molecular ecology, conservation genetics, and forensic analysis, as well as DNA bar-coding projects.     

Design of molecular markers for identification of species of the genus Chironomus (Diptera,Chironomidae)

Accurate identification and differentiation of species of the genus Chironomus based on their morphological features is a difficult problem. Unambiguous species identification by means of molecular markers is possible at any stage of the life cycle. Polymerase chain reaction (PCR) with species-specific primers was used to develop molecular markers (amplicons) for identification of Chironomus piger, Ch. dorsalis, and Ch. pseudothummi. Nucleotide sequences of the internal transcribed spacer region (ITS) of the locus coding for ribosomal RNA were used to design species-specific primers for these target species. Each of the species-specific primer pairs yielded species-specific amplicons (molecular markers) only with the DNA of target species: Ch. piger, Ch. dorsalis, and Ch. pseudothummi. Test PCRs with the DNA of eighteen Chironomus species confirmed the specificity of the primers obtained. The molecular markers produced in PCR with the designed species-specific primers permit reliable identification of Ch. piger, Ch. dorsalis, and Ch. pseudothummi and their differentiation from other species of the genus Chironomus.     

Implications of Hybridization,NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.     

Molecular identification and allopatric divergence of the white pine species in China based on the cytoplasmic DNA variation

Molecular identification of plant species may be highly related to the geographic isolation and speciation stages among species. In this study, we examined these possibilities in a group of white pines in China. We sampled 449 individuals from 60 natural populations of seven species from sect. Quinquefoliae subsect. Strobus. We sequenced four chloroplast DNA regions (around 3100 bp in length) and two mitochondrial DNAs (around 1000 bp in length). We identified 21 chlorotypes and 10 mitotypes. Both chlorotypes and mitotypes recovered from four species with long disjunction and restricted distributions in northern or northwestern China, Pinus sibirica, Pinus koraiensis, Pinus wallichiana and Pinus pumila are species-specific, suggesting that these cytoplasmic DNAs can distinguish them from the close relatives. Allopatric isolations should have contributed greatly to their genetic divergences. However, both chlorotypes and mitotypes recovered for Pinus dabeshanensis and Pinus fenzeliana distributed in southeastern and southern China are shared or closely related to those found in the widely distributed Pinus armandii. These two species may have diverged or derived from P. armandii recently. All of our findings together suggest that the discrimination power of the molecular identifications based on the cytoplasmic DNA barcodes may show variable discriminability depending on geographic isolation and speciation stages among the sampled species.     

Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.     

Isozyme Phenotypes for the Identification of Meloidogyne Species

Extensive studies during the last 20 years have demonstrated that enzyme phenotypes, especially those of esterases, are species-specific for Meloidogyne and can be used as reliable taxonomic characters for identification of most major and several minor species of this genus. Recent progress in electrophoretic procedures and advanced computer technology have made available automated electrophoretic apparati that can process very thin polyacrylamide slab gels on which the phenotypes of two or more enzymes can be revealed from the protein extract of a single Meloidogyne female. Presently, such apparati facilitate objective species identification. They also are convenient for performing routine field surveys to determine the relative distribution of major Meloidogyne species, conducting population dynamics studies in the field and in microplots, and testing the purity of greenhouse cultures.     

Identification of Dytiscus larvae by cytochrome oxidase I (COI) partial sequencing

Water beetles of the genus Dytiscus are large and conspicuous members of lentic freshwater habitats in much of the Northern Hemisphere. Of the six species found in Britain, four are relatively rare and/or endangered. Conservation of these beetles requires accurate identification during survey and monitoring, which is straightforward for adults. However, larvae can be much more abundant than adults in late spring and early summer and those of all species are morphologically similar. In this paper we describe a relatively simple molecular method, based a short fragment of the mitochondrial cytochrome oxidase I (COI) gene with species-specific sequences that can distinguish all six British Dytiscus. Sampling of larvae in a wetland containing a common (D. marginalis) and a rare (D. dimidiatus) species successfully identified 90 % of the individuals tested whereas morphology-based analysis failed to resolve them. Preliminary phylogenetic analysis based on COI sequences consistently separated D. semisulcatus from the other five species.     

Authentication of the proteins expression profiles (PEPs) identification methodology in a bloom of Karenia digitata,the most damaging harmful algal bloom causative agent in the history of Hong Kong

Identification of harmful algal bloom (HAB) causative agents makes use either of morphology-based techniques or genetic tools. These techniques are often time-consuming, labor intensive, and/or based on subjective judgment. Recently, matching with protein/peptide expression profiles (PEPs) obtained with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF–MS) has emerged as a new technique for species identification of various microorganisms. We were the first group to adopt this rapid and simple methodology for the identification of dinoflagellates (Lee et al., 2008). In the present study, we evaluated the use of PEPs from MALDI–TOF–MS for species identification of field samples obtained from a local red-tide caused by Karenia digitata. PEPs obtained from direct MALDI–TOF–MS analysis of field samples were compared to that of established monospecific reference culture. Several species-specific peaks of K. digitata were found in the reference monoculture and most of these observed peaks could be matched to the PEPs of the field samples. Matched species-specific peaks observed from the PEPs of field samples would allow rapid identification of the causative agents in corresponding and future HABs. Furthermore, as K. digitata is the most damaging dinoflagellate in Hong Kong's history, we were interested in obtaining its SSU, partial LSU rDNA and ITS sequences for future studies. In the present study, besides reporting on the ITS and rDNA sequences of K. digitata, phylogenetic tree analysis was also performed. The results showed that K. digitata fell within the Karlodinium clade and had a closer relationship with the Karlodinium species than the Karenia species.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.     

Oligonucleotide Array for Identification and Detection of Pythium Species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

Molecular marker analysis of <Emphasis Type="Italic">Cynanchum wilfordii</Emphasis> and <Emphasis Type="Italic">C. auriculatum</Emphasis> using the simple ARMS-PCR method with mismatched primers

SNPs can be used to discriminate allele-specific genotypes based on the 3′-terminal nucleotide of a primer corresponding to a specific SNP site. However, reliable discrimination between alleles in closely related species is not sufficient for molecular genetic identification. To overcome this problem, the present study evaluated the amplification refractory mutation system (ARMS)-PCR analysis method to discriminate closely related species, which utilizes specific primers that have a base pair change within three bases closest to the SNP site. The aim of this study was to develop a simple and precise DNA-based method for molecular authentication of C. wilfordii when compared with the highly similar species C. auriculatum. Molecular authentication of C. wilfordii and C. auriculatum was achieved using specific modified primers for DNA sequences of chloroplast TrnL-F and nuclear internal transcribed spacer (ITS) regions. A species-specific SNP was detected for each of the two species, and mismatched ARMS-PCR was conducted to specifically identify the SNP site. Plant samples collected from different locations were used to validate the selectively modified SNP markers, and the established method was determined to be effective. Therefore, this study provides a rapid and reliable method for specific identification of the medicinal plants, C. wilfordii and C. auriculatum.     

Diversity of Environmental Mycobacterium Isolates from Hemodialysis Water as Shown by a Multigene Sequencing Approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

Development of molecular and morphological markers to improve species-specific monitoring and systematics of Northeast Atlantic and Mediterranean skates (Rajiformes)

The Northeast Atlantic and Mediterranean skates (Rajidae) showed remarkable species diversity but with high morphological and ecological conservatism. Since skates are particularly vulnerable to the bottom trawl fishery, species-specific demographic surveys as well as studies defining life history and evolutionary traits are important in prioritising conservation programs. However, the identification of juveniles and adults of some species may be difficult using referenced guidelines and identification keys. Therefore, we attempt to develop markers for species identification through the parallel analysis of a 16S rDNA gene sequence and of several morphological characters on 135 individuals collected by trawl surveys in the Adriatic Sea and putatively assigned to six taxa. Species-specific haplotypes were defined for Raja miraletus, Raja montagui, Dipturus oxyrinchus, since a solid accordance between species boundaries and well-differentiated haplotypes was observed. Comparative analysis of 16S rDNA sequences allowed the identification of three juvenile specimens of Leucoraja circularis, a species that rarely occurs in the Adriatic Sea. On the contrary, morphological traits and haplotype distribution were largely discordant in Raja asterias and R. clavata. While all putative R. clavata individuals showed a unique haplotype (H-CLA), only 8 of 30 putative R. asterias individuals possessed a second weakly divergent haplotype (H-AST). The remaining 22 R. asterias carried the H-CLA. The multivariate analyses of morphometric and meristic characters in putative R. clavata and R. asterias revealed the clustering of individuals regardless of haplotypes. However, a bimodal distribution of R. asterias and R. clavata samples would suggest that two separated taxa might exist, both sharing the two 16S rDNA haplotypes. The haplotype distribution appeared to be significantly correlated only to the standardised disc length/total length (DL/TL) variation. Three alternative explanations may support this scenario: (i) an incomplete lineage sorting process in two morphologically yet distinct taxa; (ii) a recent hybridisation between the two taxa; (iii) the two taxa are morphologically plastic species and all considered morphological characters may be misleading in discriminating between them at all maturity stages, except for the DL/TL. However, further analyses on larger data sets and using molecular key markers (i.e. nuclear genes) will be needed to definitely resolve the status of these taxa. Molecular relationships among rajid taxa are largely consistent with systematics based on internal and external anatomical features. This multidisciplinary study contributed to defining the pattern of species diversity and abundance of rajids in the Adriatic Sea.     

Accurate identification of three cryptic species of rodents of the genus Calomys using RAPD-PCR and mitDNA RFLP markers

Calomys musculinus, Calomys laucha and Calomys venustus are cryptic species with overlapping distribution ranges. C. musculinus is the natural reservoir of Junin virus (Arenaviridae), the etiological agent of Argentine Hemorrhagic Fever. In epidemiological studies it is very important to unequivocally identify the species of individuals collected in the field in order to test virus infection. The purpose of this work was to describe molecular markers allowing a prompt and clear characterization of individuals of the three species. We studied the D-loop region of mitochondrial DNA by restriction fragment length polymorphism (RFLP). This region was amplified by PCR and the product was digested with nine restriction endonucleases (RE). Eight recognize 4 bp restriction sites (Taqα I, Tsp509 I, Aci I, Rsa I, Alu I, Nla III, Hae III and Mse I) and one recognized a 6 bp sequence (Ase I). Two of them (Aci I and Hae III) did not distinguish any of the three species. Alu I did not discriminate between C. musculinus and C. laucha, but clearly distinguished both from C. venustus. Taq I did not distinguish C. laucha from C. venustus, but differentiated both species from C. musculinus. Mse I distinguished the three species, but some of the polymorphisms of C. musculinus are very similar to C. laucha's restriction pattern. The enzyme Nla III distinguished the three species, but it is highly polymorphic within species. The enzymes Tsp509 I, Rsa I and Ase I clearly discriminated the three species, and patterns obtained with the three of them are recommended for reliable identification of individuals collected in the field. The same DNA samples were used to obtain Random Amplified Polymorphic DNAs (RAPDs) patterns. Several bands produced with primers A02, A06, A08, A09, B09 and OPA02 are species specific and could also be used for identification.     

First genetic evidence for the presence of the rumen fluke Paramphistomum epiclitum in Pakistan

More than 70 species of the Superfamily Paramphistomoidea, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes within this family have a predilection for the forestomach (rumen) or bile duct of the liver, where they may cause epithelial damage. Identification of adult Paramphistomum, Calicophoron, Gastrothylax and Fischoederius at the species level based on morphology requires specialised expertise, whereas molecular genetic marker analysis is more precise and transferable. In the present study, we performed molecular characterisation of twenty seven adult flukes collected from the forestomachs of buffalo, cattle and goats in the Punjab province of Pakistan. PCR and sequencing of the ITS-2 rDNA region revealed a single haplotype in all cases. Phylogenetic comparison of P. epiclitum ITS2-rDNA sequences with those from other Paramphistomum, Calicophoron, Gastrothylax and Fischoederius species was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for P. epiclitum identification. This work provides a validated species-specific marker of P. epiclitum and the first report of this parasite species from Pakistan. The results of this study also have implications for the diagnosis and control of rumen flukes in the region and the need for accurate species identification to understand parasite distribution and population genetics.     

The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae)

The genus Phoenix (Arecaceae) comprises 14 species distributed from Cape Verde Islands to SE Asia. It includes the economically important species Phoenix dactylifera. The paucity of differential morphological and anatomical useful characters, and interspecific hybridization, make identification of Phoenix species difficult. In this context, the development of reliable DNA markers for species and hybrid identification would be of great utility. Previous studies identified a 12 bp polymorphic chloroplast minisatellite in the trnG (GCC)-trnfM (CAU) spacer, and showed its potential for species identification in Phoenix. In this work, in order to develop an efficient DNA barcode marker for Phoenix, a longer cpDNA region (700 bp) comprising the mentioned minisatellite, and located between the psbZ and trnfM (CAU) genes, was sequenced. One hundred and thirty-six individuals, representing all Phoenix species except P. andamanensis,were analysed. The minisatellite showed 2-7 repetitions of the 12 bp motif, with 1-3 out of seven haplotypes per species. Phoenix reclinata and P. canariensis had species-specific haplotypes. Additional polymorphisms were found in the flanking regions of the minisatellite, including substitutions, indels and homopolymers. All this information allowed us to identify unambiguously eight out of the 13 species, and overall 80% of the individuals sampled. Phoenix rupicola and P. theophrasti had the same haplotype, and so had P. atlantica, P. dactylifera, and P. sylvestris (the “date palm complex” sensu Pintaud et al. 2013). For these species, additional molecular markers will be required for their unambiguous identification. The psbZ-trnfM (CAU) region therefore could be considered as a good basis for the establishment of a DNA barcoding system in Phoenix, and is potentially useful for the identification of the female parent in Phoenix hybrids.