Intraspecific Genetic Distances Research Articles

Molecular taxonomy of the two Leishmania vectors Lutzomyia umbratilis and Lutzomyia anduzei (Diptera: Psychodidae) from the Brazilian Amazon.

BackgroundLutzomyia umbratilis (a probable species complex) is the main vector of Leishmania guyanensis in the northern region of Brazil. Lutzomyia anduzei has been implicated as a secondary vector of this parasite. These species are closely related and exhibit high morphological similarity in the adult stage; therefore, they have been wrongly identified, both in the past and in the present. This shows the need for employing integrated taxonomy.MethodsWith the aim of gathering information on the molecular taxonomy and evolutionary relationships of these two vectors, 118 sequences of 663 base pairs (barcode region of the mitochondrial DNA cytochrome oxidase I – COI) were generated from 72 L. umbratilis and 46 L. anduzei individuals captured, respectively, in six and five localities of the Brazilian Amazon. The efficiency of the barcode region to differentiate the L. umbratilis lineages I and II was also evaluated. The data were analyzed using the pairwise genetic distances matrix and the Neighbor-Joining (NJ) tree, both based on the Kimura Two Parameter (K2P) evolutionary model.ResultsThe analyses resulted in 67 haplotypes: 32 for L. umbratilis and 35 for L. anduzei. The mean intra-specific genetic distance was 0.008 (0.002 to 0.010 for L. umbratilis; 0.008 to 0.014 for L. anduzei), whereas the mean interspecific genetic distance was 0.044 (0.041 to 0.046), supporting the barcoding gap. Between the L. umbratilis lineages I and II, it was 0.009 to 0.010. The NJ tree analysis strongly supported monophyletic clades for both L. umbratilis and L. anduzei, whereas the L. umbratilis lineages I and II formed two poorly supported monophyletic subclades.ConclusionsThe barcode region clearly separated the two species and may therefore constitute a valuable tool in the identification of the sand fly vectors of Leishmania in endemic leishmaniasis areas. However, the barcode region had not enough power to separate the two lineages of L. umbratilis, likely reflecting incipient species that have not yet reached the status of distinct species.

DNA barcode of Parodontidae species from the La Plata river basin - applying new data to clarify taxonomic problems

In the past years, DNA barcoding has emerged as a quick, accurate and efficient tool to identify species. Considering the difficulty in identifying some Parodontidae species from the La Plata basin and the absence of molecular data for the group, we aimed to test the effectiveness of DNA barcoding and discuss the importance of using different approaches to solve taxonomic problems. Eight species were analyzed with partial sequences of Cytochrome c oxidase I. The mean intraspecific K2P genetic distance was 0.04% compared to 4.2% for mean interspecific K2P genetic distance. The analyses of distance showed two pairs of species with K2P genetic divergence lower than 2%, but enough to separate these species. Apareiodon sp. and A. ibitiensis, considered as the same species by some authors, showed 4.2% genetic divergence, reinforcing their are different species. Samples of A. affinis from the Uruguay and Paraguay rivers presented 0.3% genetic divergence, indicating a close relationship between them. However, these samples diverged 6.1% from the samples of the upper Paraná River, indicating that the latter represents a potentially new species. The results showed the effectiveness of the DNA barcoding method in identifying the analyzed species, which, together with the morphological and cytogenetic available data, help species identification.

A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0–0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.

DNA barcoding of brown Parmeliae (Parmeliaceae) species: a molecular approach for accurate specimen identification, emphasizing species in Greenland

Warming of Arctic and alpine regions has a substantial impact on high-altitude/-latitude ecosystems. Shifting biomes due to climate change may lead to adjustments in species distributions and potential extinctions. Therefore, detailed monitoring is requisite to assess biologically meaningful shifts in community composition and species distributions. Some Arctic-alpine lichens have been shown to be particularly sensitive to climatic shifts associated with global change. However, accurate identification of lichenized fungal species remains challenging and may limit the effective use of lichens in climate change research. Given the inherent difficulties in accurate identification of lichenized fungi and the potential value of efficient identifications for bio-monitoring research, we investigated the utility of DNA barcode identification of the 13 brown Parmeliae (Ascomycota) species occurring in Greenland. For these species, we assessed monophyly and genetic distances using the nuclear ribosomal internal transcribed spacer region (ITS), the standard DNA barcode for fungi. We also compared intraspecific distance values to a proposed intra-interspecific threshold value for Parmeliaceae to identify nominal taxa potentially masking previously unrecognized diversity. Our results indicated that the 13 brown Parmeliae species occurring in Greenland can be successfully discriminated using the ITS region. All phenotypically circumscribed species were recovered as well-supported, monophyletic clades. Furthermore, our data supported a barcode gap among congeners for all brown Parmeliae species investigated here. However, high intraspecific genetic distances suggest the potential for previously unrecognized species-lineages in at least five species: Melanelia agnata, M. hepatizon, Montanelia disjuncta, M. panniformis, and M. tominii. Our research facilitates effective, long-term bio-monitoring of climate change in Greenland using lichens by providing accurate molecular identification of brown Parmeliae specimens.

Application of the ITS2 Region for Barcoding Medicinal Plants of Selaginellaceae in Pteridophyta

BackgroundSelaginellaceae is a family of nonseed plants with special evolutionary significance. Plants of the family Selaginellaceae are similarly shaped and easily confused, complicating identification via traditional methods. This study explored, for the first time, the use of the DNA barcode ITS2 to identify medicinal plants of the Selaginellaceae family.Methodology/Principal FindingsIn our study, 103 samples were collected from the main distribution areas in China; these samples represented 34 species and contained almost all of the medicinal plants of Selaginellaceae. The ITS2 region of the genome was amplified from these samples and sequenced using universal primers and reaction conditions. The success rates of the PCR amplification and sequencing were 100%. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 regions, while the presence of a barcoding gap was obvious. Using the BLAST1 and nearest distance methods, our results proved that the ITS2 regions could successfully identify the species of all Selaginellaceae samples examined. In addition, the secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Selaginellaceae. Furthermore, cluster analysis using the ITS2 barcode supported the relationship between the species of Selaginellaceae established by traditional morphological methods.ConclusionThe ITS2 barcode can effectively identify medicinal plants of Selaginellaceae. The results provide a scientific basis for the precise identification of plants of the family Selaginellaceae and the reasonable development of these resources. This study may broaden the application of DNA barcoding in the medicinal plant field and benefit phylogenetic investigations.

Genetic diversity of Clonorchis sinensis (Trematoda: Opisthorchiidae) in the Russian southern Far East based on mtDNA cox1 sequence variation

We examined the phylogeography and the variation of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit 1 gene (cox1) of the Chinese liver fluke Clonorchis sinensis (Cobbold, 1875) in two geographic localities in the Russian southern Far East and compared them with those from different geographical regions (China, Korea, Japan and Vietnam). The Russian samples differed from those of the other regions in haplotype frequencies, haplotype and nucleotide diversities, and AT/GC ratios. Only 4 of the 18 haplotypes were common to Russian and Chinese samples, and two haplotypes were common to Russia and other regions. The intraspecific genetic distances ranged from 0 to 1.58% for the entire dataset studied and from 0 to 1.25% among the samples from Russia. Phylogenetic trees revealed no significant genealogical clades of samples corresponding to sampling localities and no strong isolation by distance was estimated with Mantel test. Neutrality test analysis suggested a relatively recent population expansion for C. sinensis, whereas goodness-of-fit tests indicated deviation from the strict model of uniform expansion. Therefore, the sequences of the mtDNA cox1 gene provide useful genetic markers for evaluating intraspecific diversity and generating phylogeographic reconstructions for this fish-borne trematode.

DNA barcode identification of lichen-forming fungal species in the Rhizoplaca melanophthalma species-complex (Lecanorales, Lecanoraceae), including five new species

Recent studies using sequence data from multiple loci and coalescent-based species delimitation have revealed several species-level lineages within the phenotypically circumscribed taxon Rhizoplaca melanophthalma sensu lato. Here, we formally describe five new species within this group, R. occulta, R. parilis, R. polymorpha, R. porterii, and R. shushanii, using support from the coalescent-based species delimitation method implemented in the program Bayesian Phylogenetics and Phylogeography (BPP) as the diagnostic feature distinguishing new species. We provide a reference DNA sequence database using the ITS marker as a DNA barcode for identifying species within this complex. We also assessed intraspecific genetic distances within the six R. melanophthalma sensu lato species. While intraspecific genetic distances within the five new species were less than or equal to the lowest interspecific pairwise comparison values, an overlap in genetic distances within the R. melanophthalma sensu stricto clade suggests the potential for additional phenotypically cryptic lineages within this broadly distributed lineage. Overall, our results demonstrate the potential for accurately identifying species within the R. melanophthalma group by using molecular-based identification methods.

Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences

The DNA barcoding method was used to accurately and rapidly identify Corni Fructus and its adulterants. MethodsGenomic DNA extracted from Corni Fructus and its adulterants were used as templates. The ITS (internal trascribed spacer) regions were amplified using polymerase chain reaction. Sequence assembly was performed using CodonCode Aligner V 3.5.4. Genetic distances were computed using MEGA V 5.0. Species identification was conducted using neighbor-joining (NJ) trees. ResultsThe ITS sequence length of Corni Fructus was 659 bp. The average intra-specific genetic distance of Corni Fructus was 0.005, markedly lower than the inter-specific genetic distance between Corni Fructus and its adulterants (0.357). The ITS2 sequence length of Corni Fructus was 250 bp. No variation was found among the different samples. The interspecific genetic distance of ITS2 between Corni Fructus and its adulterants was 0.571. NJ trees and BLAST results indicated that Corni Fructus and its adulterants can be easily differentiated with monophyly. ConclusionITS/ITS2 regions can accurately and efficiently distinguish Corni Fructus and its adulterants. In addition, the results not only established the foundation for the clinical safety in the utilization of Corni Fructus, but also provided reference for molecular identification of other Chinese herbal medicine and Chinese herbal pieces.

Species delimitation in Cladonia (Ascomycota): a challenge to the DNA barcoding philosophy

The lichen-forming fungal genus Cladonia is species-rich with approximately 500 described species. The accepted barcode for fungi (ITS rDNA) often fails in identifying Cladonia spp. In order to find other markers that, in combination with the ITS rDNA region can be used for species identification in Cladonia, we studied the loci IGS rDNA, ef1α, rpb2 and cox1. A total of 782 sequences from 36 species have been analyzed. PCR amplification success rate, intraspecific and interspecific genetic distance variation, calculated using the K2P model, and the correct identification percentage (PCI) were taken into account to assess possible barcode regions. The marker showing the least intraspecific genetic distance range was cox1, followed by ITS rDNA and ef1α. Of the five studied markers only cox1 showed a barcoding gap. The rpb2 locus showed the highest PCI values, but it was the most difficult to amplify. The highest correct identification rates using blast method were obtained with rpb2.

Molecular differentiation of sheep and cattle isolates of Fasciola hepatica using RAPD-PCR

Understanding genetic structure and status of genetic variation of Fasciola hepatica isolates from different hosts, has important implications on epidemiology and effective control of fasciolosis. Random amplified polymorphic DNA (RAPD-PCR) was used to study the genetic variation of F. hepatica in sheep and cattle. DNA was extracted from adult helminthes removed from livers of each infected animal in slaughterhouse at East-Azerbaijan province, North-West of Iran. DNA template amplified by the polymerase chain reaction, using three oligonucleotide decamers with arbitrary DNA sequences as primers. RAPD patterns showed the specific but different pattern DNA patterns for each primer. The intraspecific similarity coefficient within two isolates of F. hepatica was ranged between 69 to 100%. Present findings showed that the interspecific genetic distance was higher than intraspecific genetic distances (19-47% compares to 0-19%). Pair wise similarity matrices generated from each isolates-primer combination were totaled and the similarity coefficient between strains were calculated both manually (Nei and Li method) and software analysis (Free-Tree-Freeware program). The inferred phylogenetic tree on the fingerprinting of these isolates clearly demonstrated the existence of population genetic diversity sub structuring within F. hepatica of sheep and cattle of Iran, raising interesting questions on the host specificity, epidemiology (e.g., zoonotic transmission) and ecology of this fluke. RAPD-PCR is useful for both individual identification and epidemiological investigations in endemic regions.

English

Great burdock (Arctium lappa L.) of the Compositae has economic importance in East Asian countries. In Taiwan, major cultivated areas are located in Gueilai Township, Pingtung County and Jiali Township, Tainan County. The present work was undertaken with two objectives of determining intraspecific genetic variations and studying the population dynamics of A. lappa L. In total, 23 individuals were collected from Gueilai (n = 15) and Jiali (n = 8). Chloroplast trnL-trnF intergenic spacer (IGS) and nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were subcloned and sequenced. Length variations among IGS sequences ranged from 805 to 808 bp and among ITS sequences ranged from 360 to 362 bp. Intraspecific genetic distances within IGS sequences ranged from 0 to 0.01 and within ITS sequences ranged from 0 to 0.023, which were lower than interspecific distances. All IGS and ITS sequences being separately scattered on each of the neighbor-joining trees with insignificant genetic differentiation indices (p > 0.05) suggested that both samples shared a recent common ancestor. Minimum spanning trees, Tajima’s and Fu’s parameters, and mismatch distributions implied that A. lappa L. had experienced a historical population expansion.   Key words: Compositae, Gueilai, Jiali, genetic distance, population expansion.

DNA barcodes identify marine fishes of São Paulo State, Brazil

Anthropogenic impacts are an increasing threat to the diversity of fishes, especially in areas around large urban centres, and many effective conservation actions depend on accurate species identification. Considering the utility of DNA barcoding as a global system for species identification and discovery, this study aims to assemble a DNA barcode reference sequence library for marine fishes from the coastal region of São Paulo State, Brazil. The standard 652 bp 'barcode' fragment of the cytochrome c oxidase subunit I (COI) gene was PCR amplified and bidirectionally sequenced from 678 individuals belonging to 135 species. A neighbour-joining analysis revealed that this approach can unambiguously discriminate 97% of the species surveyed. Most species exhibited low intraspecific genetic distances (0.31%), about 43-fold less than the distance among species within a genus. Four species showed higher intraspecific divergences ranging from 2.2% to 7.6%, suggesting overlooked diversity. Notably, just one species-pair exhibited barcode divergences of <1%. This library is a first step to better know the molecular diversity of marine fish species from São Paulo, providing a basis for further studies of this fauna - extending the ability to identify these species from all life stages and even fragmentary remains, setting the stage for a better understanding of interactions among species, calibrating the estimations about species composition and richness in an ecosystem, and providing tools for authenticating bioproducts and monitoring illegal species exploitation.

Identification of cortex herbs using the DNA barcode nrITS2

To discriminate the cortex herbs of Chinese Pharmacopoeia, the second internal transcribed spacer (ITS2) of ribosomal DNA of 51 samples belonging to 19 kinds of cortex herbs were analyzed in this paper. Sequence assembly was performed using the CodonCode Aligner. Phylogenetic study was performed using the molecular evolutionary genetics analysis (MEGA) 4.0 software in accordance with the Kimura 2-Parameter (K2P) model. Phylogenetic tree was constructed using the neighbor-joining (NJ) method. We found that the ITS2 sequences of the studied samples ranged from 207 to 256 bp in length and easy to be amplified. The intraspecific genetic distance of the cortex herbs was between 0 and 0.073, which was lower than their interspecific genetic distance (mean, 0.868; minimum, 0.101). In the cluster dendrogram, all the studied medicinal materials with several samples, were monophyletic. It is concluded that ITS2 barcode is suitable for the identification of cortex herbs of Chinese Pharmacopoeia, and it will play an important role in the field of identification of traditional Chinese medicine (TCM).

The molecular identification and genetic diversity of economically important wireworm species (Coleoptera: Elateridae) in Canada

Click beetle larvae (wireworms) are significant crop pests in Europe and North America. In Canada, there are ~30 economically important species which are morphologically difficult to identify, but for which sequence data are lacking. Accurate knowledge of damage-causing species and the population genetics and phylogeography of elaterids will provide insight into their sustainable management. Here, we use interspecific variation in mitochondrial 16S rRNA as a robust method of identification, consider the intra- and interspecific genetic variation of some important Canadian wireworm pests and assess the genetic structure and isolation by distance for a re-emerging major pest species, Hypnoidus bicolor Eschscholtz. Wireworms were sampled from Canada and the USA, identified as morphospecies, and sequenced at the 16S rRNA region (294–442 bp). Within some species unusually high intraspecific genetic distances between samples suggested the possibility of cryptic wireworm species or misidentifications, though this was <1 % for most species. Phylogenetic analyses gave some indication of the likely identity of these ambiguous samples. There was a significant correlation between genetic and geographic distance and significant genetic structuring within and between H. bicolor populations, which appeared to be composed of two species comprising several haplotypes. These data provide a starting point for determining the distribution of damage-causing species throughout Canada. The inclusion of data from other nuclear and mitochondrial loci, and use of sequence data from known adult samples, would further aid identification and relationships of wireworm species.

Identification of Junci Medulla using DNA barcoding technique

To identify Junci Medulla using the ITS2 barcode. The ITS2 regions of Juncus effuses and its closely related species were PCR amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance, PWG Distance and neighbor-joining (NJ) methods. The intra-specific genetic distances of J. effuses were ranged from 0 to 0.005, which were far lower than inter-specific genetic distances between J. effuses and its closely related species (0.215-0.614). All the four methods showed that ITS2 could discriminate J. effuses from its closely related species correctly. The ITS2 region is an efficient barcode for authentication of Junci Medulla, and our study further confirmed the ability of ITS2 to identify traditional Chinese medicinal materials.

Using ITS2 barcode to identify Ephedrae Herba

To identify Ephedrae Herba using the ITS2 barcode and to secure its quality and safety in medication. Total genomic DNA was isolated from Ephedrae Herba and its closely related species. Nuclear DNA ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance and neighbor-joining (NJ) methods. The intra-specific genetic distances of Ephedrae Herba were ranged from 0 to 0.002. The inter-specific genetic distances between Ephedrae Herba and its closely related species were ranged from 0.004 to 0.034. All the three methods showed that ITS2 could discriminate Ephedrae Herba from its closely related species correctly. The ITS2 region is suitable to be used for authentication of Ephedrae Herba, and our study further confirmed the effectiveness of ITS2 to identify traditional Chinese medicinal materials.

Applying DNA barcoding technique to identify Menthae Haplocalycis Herba

To identify Menthae Haplocalycis Herba and its closely related species using DNA barcoding technique. Total genomic DNA was isolated from Mentha canadensis and its closely related species. Nuclear DNA ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.0. The Kimura 2-Parameter (K2P) distances were calculated using software MEGA 5.0. Identification analyses were performed using BLAST1, Nearest Distance and neighbor-joining (NJ) methods. The intra-specific genetic distances of M. canadensis were ranged from 0 to 0.006, which were lower than inter-specific genetic distances between M. canadensis and its closely related species (0.071-0.231). All the three methods showed that ITS2 could discriminate M. canadensis from its closely related species correctly. The ITS2 region is an efficient barcode for identification of Menthae Haplocalycis Herba, which provides a scientific basis for fast and accurate identification of the herb.

Molecular identification of Cistanches Herba and its adulterants based on nrITS2 sequence

Cistanches Herba is a commonly used Chinese medicine for its tonification properties. However, it is often confused and substituted with the dried succulent stems of Orobanche pycnostachya, Orobanche coerulescens, Boschniakia rossica, Cistanche salsa, Cistanche sinensis and Cynomorium songaricum. Identifying Cistanches Herba based only on its morphological and chemical appearance is generally difficult. To develop a convenient and efficient identification method for discriminating Cistanches Herba and its adulterants, the second internal transcribed spacer (ITS2) of the ribosomal DNA of 39 individuals from Cistanches Herba and its adulterants were examined in this study. A phylogenetic tree was constructed using the neighbor-joining (NJ) and Kimura 2-parameter (K2P) methods. The results showed that the length of ITS2 sequence of Cistanche deserticola and Cistanche tubulosa were 229 and 233 bp, respectively. The intraspecific genetic distance of the two botanical origins of Cistanche Herba were both lower than their respective interspecific genetic distance with their adulterants. In the cluster dendrogram, C. deserticola and C. tubulosa were both monophyletic, and were distinguished from their adulterants. In conclusion, ITS2 can be used as a DNA barcode to efficiently identify Cistanches Herba and its adulterants, and can provide important information for quality evaluation, resource protection, exploitation, and sustainable utilization of this medicinal material. Key words: Cistanches Herba, Orobanchaceae, internal transcribed spacer (ITS2), DNA barcode, molecular Identification.

Genetic diversity of &lt;i&gt;Neosalanx taihuensis&lt;/i&gt; based on mitochondrial COI sequences

利用线粒体细胞色素C氧化酶I(COI)分子标记分析长江中下游太湖新银鱼(Neosalanx taihuensis)8个地理种群132个样本的遗传多样性.该基因630 bp片段的碱基序列共检出8个核苷酸变异位点(变异率1.27%),其中局域性单倍型居多(75%),群体单倍型多样性较高(h=0.576±0.036),而核苷酸多样性较低(π=0.00112±0.00204).不同地理种群遗传多样性差异显著:有人工移植历史种群遗传多样性较高、隔离度较高的种群遗传多样性较低,但大部分的遗传变异来自于种群内(54.83%),反映出地理隔离和人为干扰对太湖新银鱼遗传格局影响显著.研究表明COI基因适于银鱼科鱼类物种鉴别和系统发育研究,同时可为同种种群间遗传关系分析提供一定的信息.;Neosalanx taihuensis is an annual fish with commercial importance. This species is endemic to China, occurring in inland waters affiliated to the eastern-flowing rivers southern to the Yellow River, e. g. the rivers of Yellow, Huaihe, Yangtze, Ou and Pearl etc. Among these river basins, the Yangtze River tests to be the most important region for this species with the highest fishery stocks and the most populations, and thus to be the core area for its conservation and sustainable exploitation. In the near decades, however, N. taihuensis decreased severely with most of the natural populations extinguished economically or even ecologically. The local population extinction infers that the species' genetic diversity must have suffered bad influence and some genetic types must have lost as result. To test this presumption, we conducted an icefish-specific investigation in the mid-lower reaches of Yangtze River during August, 2006. Totally eight populations were sampled and used in the followed experiment on their genetic diversity. Based on the gene diversity of mitochondrial cytochrome oxidase subunit I(COI), we mainly focus on two points:1) to find out the genetic diversity in single population and among populations for understanding the genetic status of wild N. taihuensis in the river basin, 2) to check the suitability of COI gene for DNA barcoding study in the family of Salangidae. We selected 132 specimens, representing eight different geographic populations in the mid-lower Yangtze River basin, and analyzed their base sequences of COI gene. Among the 630 bp nucleotide sequence of COI gene, eight(1.27%) was variation sites, contributing to eight gene haplotypes. Although the number of gene haplotype and the nucleotide diversity index(π=0.00112 ± 0.00204) were not high, haplotype diversity(h) was fairly rich(h=0.576 ± 0.036) in the populations at river basin scale. Taking spatial distribution pattern into account, most gene haplotypes distributed in spatially narrow region with quite low occurrence incidences of 12.5% or 25.0%, occurred in one or two lakes respectively. Haplotype richness in each population varied significantly from each other, with a high fixation index. The least haplotypes occurred in the two highly isolated lakes of Dongting and Liangzi, and the highest haplotype richness occurred in the two lakes with icefish introduction histories. This result indicates that a high inter-population variation existed in the studied region. So we concluded that wild populations of N. taihuensis have suffered great ecological isolation, although they are geographically near to each other. Comparing with several other salangid fishes, e. g. Protosalanx hyalocranius, Hemisalanx brachyrostralis and N. oligodontis, the inter-specific genetic distance was 4.443%, amounting to some 35 times of that in N. taihuensis populations(0.125%). Compared with other fish taxa, such as Coilia, Culter, Oncorhynchus and Salmo, the intra-specific genetic distance was much smaller within N. taihuensis populations(< 1%). In conclusion, the COI gene is good and suitable to DNA barcode study in species identifying and systematic study, and can be useful in the assessment, monitoring and conservation management of icefish genetic diversity.

RAPD–SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae)

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.