Species identification of sympatric cuckoo nestlings in a multiple-cuckoo system,China

Interaction between a parasite and its host could lead to a co-evolutionary arms race. Cuckoo-host system is among the most studied of all brood parasite systems, but the cuckoos of Asia, on the other hand, are much less well known. China has the most abundant cuckoo species in Asia. Many of these co-occur in sympatric areas, posing a potential risk of mis-identification of cuckoo nestlings, especially in Cuculus species. In this study we have provided a practical criterion to identify cuckoo nestlings species in the field and performed molecular phylogeny to confirm our empirical results. These results indicate that two distinct characteristics of cuckoo nestlings, i.e., the gape color pattern and feather traits can be considered as reliable species identification. To our knowledge, this is the first report for species identification of Cuculus nestlings through molecular analysis.

Wood species identification using spectral reflectance feature and optimal illumination radian design

We developed a scheme based on wood surface novel wood recognition spectral features that aimed to solve three problems. First was elimination of noise in some bands of wood spectral reflection curves. Second was improvement of wood feature selection based on analysis of wood spectral data. The wood spectral band is 350-2500 nm, a 2150D vector with a spectral sampling interval of 1 nm. We developed a feature selection proce- dure and a filtering procedure by solving the eigenvalues of the dispersion matrix. Third, we optimized the design for the indoor radian＇s mounting height. We used a genetic algorithm to solve the optimal radian＇s height so that the spectral reflection curves had the best classification infor- mation for wood species. Experiments on fivecommon wood species in northeast China showed overall recogni- tion accuracy 〉95 % at optimal recognition velocity.

Molecular genetic strategies for species identification

This paper probes into the molecular genetic mechanism of the formation of species, subspecies and variety in evolving progression, and brings forward 5 criteria of an ideal strategy in species identification: stating the specific characteristics at species, subspecies and variety level without any interference of too high polymorphism at individual or population level; keys should be distributed as 0 or 1, e. g. yes or no; satisfying repeatability and simple operation; high veracity and reliability; adaptability to widely various specimen. Respectively, this paper reviews two strategies focusing on detecting the fragment length polymorphism and base replacement and lays out some detail methods under above strategies. It demonstrates that it is not possible to solve all species problems by pursuing identification with only a single gene or DNA fragment. Only based on thorough consideration of all strategies, a method or combined several methods could bring satisfying reliability. For advanced focuses, it requires not only development and optimization of methods under above strategies, but also new originality of creative strategies.

Species identification and phylogenetic analysis of genus Nassarius(Nassariidae)based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.

Species identification of crested gibbons(Nomascus) in captivity in China using karyotyping-and PCR-based approaches

Gibbons and siamangs （Hylobatidae） are well-known for their rapid chromosomal evolution,which has resulted in high speciation rate within the family.On the other hand,distinct karyotypes do not prevent speciation,allowing interbreeding between individuals in captivity,and the unwanted hybrids are ethically problematic as all gibbon species are endangered or critically endangered.Thus,accurate species identification is crucial for captive breeding,particularly in China where studbooks are unavailable.Identification based on external morphology is difficult,especially for hybrids,because species are usually similar in appearance.In this study,we employed G-banding karyotyping and fluorescence in situ hybridization （FISH） as well as a PCR-based approach to examine karyotypic characteristics and identify crested gibbons of the genus Nomascus from zoos and nature reserves in China.We characterized and identified five karyotypes from 21 individuals of Nomascus.Using karyotypes and mitochondrial and nuclear genes,we identified three purebred species and three hybrids,including one F2 hybrid between N.gabriellae and N.siki.Our results also supported that N.leucogenys and N.siki shared the same inversion on chromosome 7,which resolves arguments from previous studies.Our results demonstrated that both karyotyping and DNA-based approaches were suitable for identifying purebred species,though neither was ideal for hybrid identification.The advantages and disadvantages of both approaches are discussed.Our results further highlight the importance of animal ethics and welfare,which are critical for endangered species in captivity.

Progress of DNA-based Methods for Species Identification

Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The current review shows common target genes and screening criteria suitable for species identification, and described various DNA-based molecular biology methods about species identification. Additionally, it discusses the future development of species identification combined with real-time PCR and sequencing technologies.

Molecular identification of scallop planktonic larvae using species-specific microsatellites

The identification of scallop larvae is essential to understand the population structure and community dynamics and to assess the potential environmental impacts caused by scallop larvae released or escaped. However, the larvae identification by morphological characteristics is notoriously difficult, mainly due to the small size （usually being less than 150 μm） and vague morphological characteristics among different scallop species. A simple and accurate molecular method was developed to identify four economically farmed scallop species, the Zhikong scallop Chlamys farreri, the noble scallop C. nobilis, the bay scallop Argopecten irradians and the Yesso scallop Mizuhopecten yessoensis. The tests used the high degree of species-specific microsatellite markers, which was specified by transferability analyses, assessed by reference individuals and evaluated by BLAST searches. The sensitivity test indicated that the species-specific microsatellites were sensitive enough for the detection of 1% -2% larvae in mixed plankton samples. Larvae collected from scallop hatcheries and their effluents and from the artificially controlled crosses were well identified to the species/hybrid level. The results demonstrated that the one-step PCR-based assay was technically simple, inexpensive and robust in identification analyses, and also less sensitive to initial quality of template DNA extracted from the ethanol-preserved samples for several years.

Identification of Five Sea Cucumber Species Through PCR-RFLP Analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene(COI) was used to identifing five sea cucumber species(Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31 I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

Identification of Rhodiola species by using RP-HPLC

An approach was established using RP-HPLC (reversed-phase high-performance liquid chromatography) to identify ten species of Rhodiola, R. coccinea A. Bor, R. junggarica C.Y. Yang et N.R. Cui spn., R. heterodonta A. Bor, R. linearifolia A. Bor, R. pamiro alaiucm A. Bor, R. kaschgarica A. Bor, R. litwinowii A. Bor, R. gelida schrenk, R. rosea L. and R. quadrifide Fisch et Mey collected from the Tianshan Mountains areas of China. Chromatograms of alcohol-soluble proteins, generated from these ten Rhodiola spp. were compared. Each chromatogram of alcohol-soluble proteins came from a single seed of one wild species only. The results showed that when using a Waters Delta Pak. C18, 5 μm particle size reversed phase column (150 mm×3.9 mm), a linear gradient of 22%?55% solvent B with a flow rate of 1 ml/min and a run time of 67 min, the chromatography gave optimum separation of Rhodiola alcohol-soluble proteins. Chromatogram of each species was different and could be used to identify those species. Cluster analysis of genetic similarity coefficients of 37% to 60% showed a medium degree of genetic diversity among the species in these eco-areas. Cluster analysis showed that the ten species of Rhodiola can be divided into four clusters and yielded the general and unique biochemical markers of these species. RP-HPLC was shown to be a rapid, repeatable and reliable method for Rhodiola species identification and analysis of genetic diversity.

Identification of Kalidium species(Chenopodiaceae)by DNA barcoding

DNA barcoding is an increasingly prevalent molecular biological technology which uses a short and conserved DNA fragment to facilitate rapid and accurate species identification. Kalidium species are distributed in saline soil habitat throughout Southeast Europe and Northwest Asia, and used mainly as forage grass in China. The discrimination of Ka-lidium species was based only on morphology-based identification systems and limited to recognized species. Here, we tested four DNA candidate loci, one nuclear locus （ITS, internal transcribed spacer） and three plastid loci （rbcL9 matK and ycflb）, to select potential DNA barcodes for identifying different Kalidium species. Results showed that the best DNA barcode was ITS locus, which displayed the highest species discrimination rate （100%）, followed by matK （33.3%）,ycflb （16.7%）, and rbcL （16.7%）. Meanwhile, four loci clearly identified the variant species, Kalidium cuspidatum （Ung.-Stemb.） Gmb.var.A. J. Li,as a single species in Kalidium.

Establish an allele-specific real-time PCR for Leishmania species identification

Background:Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.Methods:A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.Results:This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.Conclusions:Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.

Screening of Species-specific DNA Probes for Identification of Fallopia multiflora

To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA （gDNA） suppression subtraction hybridization （SSH） between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species （adulterants and/or closely related species of F. muhiflora） and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora.

A Comparative Study of Human and Animal Hairs:Microscopic Hair Comparison and Cytochrome c Oxidase I Species Identification

Human and animal hairs have been used in forensic investigations for over a century.Hair is stable under adverse natural conditions;hence,it is often recovered at the crime scene,and it is necessary to determine whether the hair is of human or animal origin.Morphological and genetic characteristics are useful to differentiate human hair from animal hair.In the present study,we analyzed the distinguishing characteristics of hair of various species.In addition,we explore species identification by cytochrome c oxidase I mitochondrial gene analysis.We confirm that both the microscopic and molecular analyses of hairs are useful in forensic investigations.

Plastid phylogenomics and species discrimination in the“Chinese”clade of Roscoea(Zingiberaceae)

Roscoea is an alpine or subalpine genus from the pan-tropical family Zingiberaceae,which consists of two disjunct groups in geography,namely the"Chinese"clade and the"Himalayan"clade.Despite extensive research on the genus,Roscoea species remain poorly defined and relationships between these species are not well resolved.In this study,we used plastid genomes of nine species and one variety to resolve phylogenetic relationships within the"Chinese"clade of Roscoea and as DNA super barcodes for species discrimination.We found that Roscoea plastid genomes ranged in length from 163,063 to 163,796 bp,and encoded 113 genes,including 79 protein-coding genes,30 tRNA genes,four rRNA genes.In addition,expansion and contraction of the IR regions showed obvious infraspecifc conservatism and interspecific differentiation.Plastid phylogenomics revealed that species belonging to the"Chinese"clade of Roscoea can be divided into four distinct subclades.Furthermore,our analysis supported the independence of R.cautleoides var.pubescens,the recovery of Roscoea pubescens Z.Y.Zhu,and a close relationship between R.humeana and R.cautloides.When we used the plastid genome as a super barcode,we found that it possessed strong discriminatory power(90%)with high support values.Intergenic regions provided similar resolution,which was much better than that of protein-coding regions,hypervariable regions,and DNA universal barcodes.However,plastid genomes could not completely resolve Roscoea phylogeny or definitively discriminate species.These limitations are likely related to the complex history of Roscoea speciation,poorly defined species within the genus,and the maternal inheritance of plastid genomes.

Novel Species Including Mycobacterium fukienense sp. Is Found from Tuberculosis Patients in Fujian Province, China, Using Phylogenetic Analysis of Mycobacterium chelonae/abscessus Complex

Objective To identify the novel species ‘Mycobacterium fukienense＇ sp. nov of Mycobacterium chelonoe/abscessus complex from tuberculosis patients in Fujian Province, China. Methods Five of 27 clinical Mycobucterium isolates （CIs） were previously identified as M. chelonoe/obscessus complex by sequencing the hsp65, rpoB, 165-235 rRNA internal transcribed spacer region （its）, recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobocterium. Clinical Mycobecterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. Results The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 165-235 rRNA internal tronscribed spacer region （its）, sodA, and recA genes as compared with the M. obscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. Conclusion The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. cheloneo/abscessus complex.

Development of novel microsatellite markers for Holothurian scabra (Holothuriidae), Apostichopusjaponicas (Stichopodidae) and cross-species testing in other sea cucumbers

Thirty-five new microsatellite loci from the sea cucumbers Holothurian scabra （Jaeger, 1833） and Apostichopus japonicas （Selenka, 1867） were screened and characterized using the method of magnetic bead enrichment. Of the twenty-four polymorphic loci tested, eighteen were consistent with Hardy-Weinberg equilibrium after a modified false discovery rate （B-Y FDR） correction, whereas six showed statistically significant deviations （CHS2 and CHSll： P〈0.014 790; FCS1, FCS6, FCS8 and FCS14：P〈0.015 377）. Furthermore, four species ofplesiomorphous and related sea cucumbers （Holothurian scabra, Holothuria leucospilota, Stichopus horrens and Apostichopus japonicas） were tested for mutual cross-amplification using a total of ninety microsatellite loci. Although transferability and universality of all loci were generally low, the results of the cross-species study showed that the markers can be applied to identify individuals to species according to the presence or absence of specific microsatellite alleles. The microsatellite markers reported here will contribute to the study of genetic diversity, assisted breeding, and population conservation in sea cucumbers, as well as allow for the identification of individuals to closely related species,

Molecular Identification of Dried Shellfish Products Sold on the Market Using DNA Barcoding

The dried shellfish products with rich nutrients and low-calorie content are favorite food in China,especially in coastal areas.However,the species of dried shellfish products in the market are usually unknown,as the taxonomic features were removed during the production process.This study described the application of DNA barcoding technique to the identification of 100 dried shellfish(scallop,squid,octopus and cuttlefish)products in markets.Samples were authenticated by comparing mitochondrial cytochrome oxidase subunit I(COI)gene and 16S ribosomal RNA(16S rRNA)gene sequences with public reference taxonomic databases.The results showed that all the 100 products can be identified at species level.Sixty four scallop adductor products were processed using the bay scallop,Argopecten irradians,and one was from Portuguese oyster,Crassostrea angulata.All the 27 squid,2 cuttlefish and 6 octopus products were produced by the Jumbo flying squid,Dosidicus gigas.The neighbour-joining tree is in agreement with the results of DNA barcoding analysis.The 64 scallop samples formed one A.irradians cluster,leaving Sca65 clustered with the reference oyster sequence C.angulata(MH997922).All the 35 cephalopod(squid,octopus and cuttlefish)samples formed a D.gigas cluster.This investigation revealed a low variety of dried shellfish products sold on the market,and highlighted the high rate of mislabeling and species substitution.Our present work provides a practical method for tracing and authenticating shellfish products.

Development and evaluation of a novel multiplex probe array for rapid differential identification of Mycobacterium in clinical specimens

Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex- specific probe. There were 79.3 % （23/29） of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe army will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.

Application of Molecular Marker Technology in the Study of Forest Tree Species

Due to its unique advantages, molecular marker technology is widely applied in the research of forest tree species. This paper reviewed the application of molecular marker technology in tree species resource diversity, germplasm identification, genetic map construction, gene mapping and marker-assisted selection (MAS) breeding. In addition, it elaborated the great significance of molecular marker technology to promote the sustainable development of forestry production in China.

Evaluating the effectiveness of DNA barcoding for genetic identification of Canthocamptidae (Harpacticoida, Copepoda) of Lake Baikal

Ancient Lake Baikal(LB)is the prime hotspot of the harpacticoid diversity.The Canthocamptidae harpacticoids in the lake can be considered at least six species flocks.The lacks of unequivocal diagnostic characters in the family species description often lead species identification to an impasse.Recently developed molecular techniques provide a powerful tool to subject copepod taxa to comparative analyses.In order to provide a new base for canthocamp-tids taxonomy and clarify the systematic in some species flocks,we analyzed the mitochondrial DNA marker COI of 103 harpacticoid specimens from LB belonging to five genera.The materials were obtained from different lake basins from the depths 0.2-1632 m.Using DNA barcoding,we identified 23 genetic species or groups of species,it was almost 1.5 times less than number of morphological species,and divergence of some molecular lineages were not corresponding to morphological species.Results of our study were the discovery of a new species,males of some morphological species,for which only females were previously described,clarification of the level of morphological variability of some species,etc.Specifically,it became apparent that morphological and genetic harpacticoid species diversity in LB differ significantly and it has to be detected strictly in complex.