Intraspecies Genetic Distance Research Articles

DNA barcoding of Burgo chicken from Bengkulu, Indonesia, based on the cytochrome oxidase gene sub unit I mitochondria DNA

Abstract. Utama AF, Jarulis, Sipriyadi, Jannah IM. 2023. DNA barcoding of Burgo chicken from Bengkulu, Indonesia, based on the cytochrome oxidase gene sub unit I mitochondria DNA. Biodiversitas 24: 6268-6275. The Burgo chicken provides a genetic source of one crossbreed chicken in Bengkulu Province. However, genetic information on Burgo chickens is not yet available, while the population of this chicken continues to decline. Research on DNA barcoding using the cytochrome oxidase subunit I mtDNA gene was conducted to obtain DNA barcodes and the relationship between Burgo chickens and other chicken species based on the COI mtDNA gene. Blood samples were obtained from Burgo chickens belonging to the Bengkulu Burgo chicken hobbyist community. Collected blood was isolated following the dneasy® blood and tissue kit protocol based on the spin-column protocol procedure, Qiagen. Amplification of genomic DNA using Polymerase Chain Reaction. The product was electrophoresed on a 1.2% agarose gel and visualized under UV light using Gel Documentation System, Axygen. Samples with bright DNA bands proceeded to the sequencing process. Sequencing results were analyzed using MEGA 11.0 software. The results showed that the target length of the Burgo chicken band was in accordance with the primer design used (752pb). SNPs were obtained at 10 specific sites in Burgo chickens and had a species barcode at site 746. The intraspecies genetic distance was 0.6%, interspecies 1.2%, and with the outgroup 14.5%, there were 12 haplotypes from all samples (n=15). Based on the result, we can conclude that the Burgo chicken is closely related to the red jungle fowl compared to other chicken species.

Appressoria-Producing Sordariomycetes Taxa Associated with Jasminum Species.

Appressoria are specialized structures formed by certain phytopathogenic fungi during the early stages of the infection process. Over the years, significant advancements have been made in understanding the formation, types, and functions of appressoria. Besides being formed primarily by fungal pathogens, many studies have reported their occurrence in other life modes such as endophytes, epiphytes, and saprobes. In this study, we observed the formation of appressoria in fungal genera that have been found associated with leaf spots and, interestingly, by a saprobic species. We used morphological descriptions and illustrations, molecular phylogeny, coalescent-based Poisson tree processes (PTP) model, inter- and intra-species genetic distances based on their respective DNA markers, and Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR) to establish a new species (Pseudoplagiostoma jasmini), a Ciliochorella sp., and a new host record (Coniella malaysiana). The Ciliochorella sp. is reported as a saprobe, while Pseudoplagiostoma jasmini and Coniella malaysiana were found to be associated with leaf spots of Jasminum species. All three taxa produce appressoria, and this is the first study that reports the formation of appressoria by a Ciliochorella sp. and a Pseudoplagiostoma sp.

New Record of Champsodon Atridorsalis Ochiai & Nakamura, 1964 from the South Eastern Arabian Sea

The present study reports a new record of Champsodon atridorsalis from the Arabian Sea along the Indian coast. Two specimens were obtained from the experimental fishing conducted in F V Silver pompano off Alappuzha coast Kerala, southwest coast of India in October 2018. The species was observed to have a snout length greater than the eye diameter; premaxilla with a shallow notch lateral to symphysis; maxilla extending beyond the posterior margin of the eye; small fleshy tabs of skin in the lower fold of the skin of the eye. The intraspecies genetic distance in the 16 S gene of Champsodon atridorsalis retrieved from NCBI revealed 0.0% from Indian waters, while it showed 0.15% with the species recovered from the USA, confirming the species. The cluster analysis formed a single group showing homogeneity, and a sequence of Sardinella longiceps (KX590730) was used as an outgroup.

Testing and using complete plastomes for authentication of medicinal Polygonatum species (Asparagaceae)

Polygonati Rhizoma, a traditional Chinese medicine important to the pharmaceutical and food industries, is frequently adulterated with morphologically similar congeneric species, posing quality and effectiveness issues for the products. Accordingly, the accurate and sensitive authentication of medicinal Polygonatum species is vital to the standardization of Polygonati Rhizoma products. In view of the difficulties in morphological analysis and the use of standard DNA barcodes for authentication of medicinal Polygonatum species, an alternative authentication method is needed. Based on inter- and intraspecies sampling, phylogenetic and genetic distance analyses were performed to evaluate the discriminative power of complete plastomes for the identification of Polygonatum species. The results indicate that the genetic criteria of complete plastomes are in good correspondence with species boundaries and therefore can be used for unambiguous identification of medicinal Polygonatum species and morphologically similar congeners, which verifies the theoretical prediction that the application of genome skimming to generate complete plastomes as molecular tags can serve as an effective and accurate method to detect whether Polygonati Rhizoma is adulterated with congeneric species. Additionally, in this study, a taxonomically well-curated and high-resolution reference plastome superbarcode library for Polygonatum was developed for the first time, providing a valuable genetic resource for the application of genome skimming to monitor and control the production and trade of Polygonati Rhizoma to guarantee the effectiveness of downstream products. The findings also provide new insights to clarify the taxonomic ambiguities of the generic circumscription of Polygonatum and Heteropolygonatum, as well as the species delimitation of Polygonatum cyrtonema, Polygonatum nodosum, and Polygonatum punctatum, which will contribute to proper exploitation and conservation of Polygonatum species.

DNA Barcoding of Key Storage Pests Using Mitochondrial Cytochrome Oxidase I

Stored grain insect pests are of economic importance, and they spread rapidly through the grain trade. Hence, it is important to identify these pests accurately. Although several morphological methods exist, it is tedious and time-consuming. DNA barcoding using mitochondrial COI is an alternative approach that aids in precise species identification. In this study, 13 stored grain insect pests belonging to the order Coleoptera and Lepidoptera were collected from different storage structures in Coimbatore. A fragment of mt-COI was amplified and sequenced. Sequence analyses were carried out with BOLD (Barcode of Life Data System) and ABGD (Automatic Barcode Gap Discovery) tools. The barcoding gap analysis revealed that the inter-species genetic distance is greater than the intra-species genetic distance. The ABGD analysis for species delimitation partitioned the Coleopteran and Lepidopteran datasets into 9 and 4 putative species, respectively. The barcode gap was absent in more closely related species. However, analysis of their sequences revealed significant variations. Our results showed the ability of the mt-COI to discriminate between the species, thus provide a complementary technique for the diagnosis of stored grain insect pests.

Integrative taxonomy-based discovery of Dussumieria modakandai sp. nov. from India.

This study is based on integrative taxonomy and reports a new fish species Dussumieria modakandai sp. nov. from India. The new species differs from three valid species within the genus by a combination of characters such as longer maxilla (9.1%-9.9% standard length vs. 8.7% in Dussumieria elopsoides, 6.3%-8.5% in Dussumieria acuta) and one or two rows of small conical teeth on palatine (vs. several rows in D. elopsoides and Dussumieria albulina). It also differs by the absence of longitudinal striae on the posterior side of body scales (vs. presence in D. acuta and D. albulina) and the absence of parasphenoid teeth (vs. presence in D. acuta). The maxilla length of D. modakandai sp. nov. is greater than snout length, which distinguishes it from other congeners. The multivariate analysis of morphometric characters using PCA differentiated the new species from D. elopsoides and D. acuta samples collected in this study. The molecular analysis, based on cytochrome c oxidase I, distinguished the new species from D. acuta, D. albulina and D. elopsoides with a high genetic distance of 13.73%, 12.22% and 12.74%, respectively. The maximum-likelihood phylogenetic tree and automatic barcode gap discovery analysis showed the existence of six putative species in Dussumieria. Even the exhaustive sub-clade formation within species and high intra-species genetic distance in D. acuta (1.59) and D. modakandai (1.95) indicate the possibility of a few more cryptic species. This warrants comprehensive sample collection across the distribution range and integrative taxonomic study of the genus Dussumieria.

DNA barcoding of deep-sea shrimp Sicyonia parajaponica Crosnier, 2003 (Crustacea: Decapoda: Sicyoniidae) from southwest coast of India.

The present work reports detailed taxonomic information on deep-sea shrimp Sicyonia parajaponica from southwest Indian waters. The samples were caught in bottom trawls conducted between the depths of 200 and 230 m from Sakthikulangara fishing harbour off Kollam, Kerala along the Arabian Sea during November 2019. DNA barcoding and a phylogenetic analysis was used to explore the relationship of the genus Sicyonia based on mitochondrial cytochrome c oxidase subunit 1 (COI: MN816389, MN816390) with the present specimen and sequences retrieved from NCBI GenBank. There is no difference in the intraspecies genetic distance (0%) while interspecies genetic distance (19.028.1%) revealed divergence between the species (COI) of the genus Sicyonia.

Genetic diversity of the Pegu Rice Frog, Microhyla berdmorei (Anura: Microhylidae) based on mitochondrial DNA

The Pegu Rice Frog, Microhyla berdmorei is distributed across ten Asian countries. However, the DNA barcoding information (COI gene) is restricted to only Southeast Asian countries. Here, we sampled a specimen of M. berdmorei in Mizoram state, northeast India to allow the genetic diversity of the species across its range. We generated both COI and 16S ribosomal RNA sequences of the studied species to check the population genetic diversity. The Bayesian analyses clearly discriminate M. berdmorei from its sister species Microhyla pulchra. The present datasets of M. berdmorei also revealed 11 and 19 haplotypes with high uncorrected pairwise genetic distances in COI (3.8–11.8%) and 16S rRNA (0–4.6%) gene, respectively. Owing to the high intra-species genetic distances and different haplotypes with sufficient mutational steps in both mitochondrial genes, this study affirms the existence of M. berdmorei species complex or cryptic diversity within its range distribution in South and Southeast Asia.

Molecular and Otolith Shape Analyses of Scorpaena spp. in the Turkish Seas

This study aims to investigate the discrimination and the phylogenetic relationship of five Scorpaena species (S. elongata, S. maderensis, S. notata, S. porcus, and S. scrofa), sampled from eight locations in the four seas (Mediterranean Sea, Aegean Sea, Black Sea, and Sea of Marmara) in Turkey, using both molecular and otolith shape analyses. Totally, 1360 samples were examined for otolith shape analyses, and 51 of them were also used in the molecular analysis (COI sequences, 652 bp). Eighteen haplotypes were determined in the 5 species of the genus Scorpaena; 3 haplotypes for S. elongata, S. maderensis and S. scrofa, 4 haplotypes for S. notata, and 5 haplotypes for S. porcus. The highest interspecific genetic distance was observed between S. maderensis and S. notata (0.1569), and the lowest interspecific genetic distance (0.0527) was observed between S. elongata and S. scrofa. The lowest interspecies genetic distance was found to be considerably higher than the highest intraspecies genetic distances for these five Scorpaena species. The barcode range in the study alone provided a high rate of barcoding success in identifying species belonging to the genus Scorpaena. A Principal Component Analysis (PCA) and Canonical Discriminant Analysis (CDA) were conducted for interspecific discrimination of the five Scorpaena species. The CDA produced an overall classification success rate of 96.3% with the highest rate for S. scrofa (98.8%), followed by S. maderensis (97.5%), S. notata 96.6%), S. elongata (94.6%), and with the lowest rate for S. porcus (94.4%). The results indicated that molecular analysis was generally compatible with otolith shape analysis and confirmed that otoliths have phylogenetic signals for Scorpaena species. This is the first study in which five Scorpaena species sampled from eight locations in four seas have been distinguished using both otolith shape and molecular analyses.

Identification and Genetic Relationship Analysis of <i>Torreya</i> Based on nrDNA ITS Sequence

In order to evaluate the identification ability of nrDNA ITS sequences on Torreya , this study used ITS sequence and four different analysis methods (BLAST, K2P genetic distance, SNP analysis, NJ tree) to identify species of Torreya , and established phylogenetic trees to discuss their phylogenetic relationships. The results showed that the length of 48 ITS sequences was 1 095 bp~1 105 bp, the average intraspecies genetic distance was 0.001 6 and the average interspecies genetic distance was 0.015 0. At the species level, BLAST alignment and NJ tree had the highest efficiency in Torreya . The SNP loci analysis could effectively identify T. grandis cv . ‘ Merrillii ’ and T. grandis . The analysis of genetic relationship showed that all the other species of Torreya were single-line branches, but the quince tree of T. yunnanensis and T. fargesii were clustered into a cluster in the ML tree, indicating that the two were closely related. This provided evidence for the nuclear genome sequence proposed the incorporation of T. yunnanensis and T. fargesii . This study showed that ITS sequence could be used as a DNA barcode for the identification of Torreya , and providing reference for the identification and systematic relationship of Torreya.

DNA barcoding of some common coastal fishes from Japan and China

To determine the species status of some common coastal fishes from Japan and China, 652 bp fragments of cytochrome oxidase subunit I (COI) of 184 individuals of 37 species, 34 genera, 24 families, 7 orders in Japan were sequenced, combined with 143 homologous sequences from Japan and China downloaded from GenBank, a total of 327 sequences of 40 species, 35 genera, 24 families and 7 orders were analyzed. Results showed that the average interspecific genetic distance (6.25%) was about 14.88 times the mean intraspecific distance (0.42%), and 36 species were monophyletic with 97%-99% bootstrap support, supporting their species status. The intraspecies genetic distance of <italic>Lateolabrax</italic> spp. was 3.91%, and formed two clades on the neighboring tree, and the genetic distances between the clades (6.98%) were about 14.2 times those within clades (0.49%), suggesting that <italic>L</italic>. <italic>japonicus</italic> from Japan and <italic>L</italic>. <italic>maculatus</italic> from China might have evolved to two different species, which was consistent with previous studies. The intraspecies genetic distance of <italic>Pennahia argentata</italic> was 2.01%, and it formed two clades with 99% bootstrap support, and the genetic distances between the clades (3.28%) were about 11.3 times those within clades (0.29%), suggesting that <italic>P</italic>. <italic>argentata</italic> from Japan and China might be 2 subspecies or 2 species. <italic>Cleisthenes herzensteini</italic> and <italic>C</italic>. <italic>Pinetorum</italic> were mixed on a clade and both average genetic distances between species (0.32%) and within species (0.23%) was less than 2%, thus it is assumed that there might be misidentification, but the possibility of incomplete lineage sorting can not be ruled out. <italic>Callionymus valenciennei</italic> and <italic>Repomucenus ornatipinnis</italic> clustered into 2 clades, and the average genetic distance between clades (1.97%) was 4.6 times that within clades (0.43%), it is therefore speculated that they might be 2 subspecies or 2 species. Due to the small size of Callionymid fish and significant morphological differences between males and females, the possibility of morphological misidentification can not be ruled out.

Genetic analysis of Carasobarbus Karaman, 1971 (Actinopterygii Cyprinidae) in Anatolia

We sequenced the complete mitochondrial cytochrome b gene (1141 bp) of 70 individuals from ten populations of three Carasobarbus species in Anatolia. Totally eight haplotypes were identified. The intraspecies genetic distance ranged from 0.00% to 0.21%, while it varied from 2.6% to 9.0% for interspecies. Except for Gaziantep samples with low sample size, high haplotype diversity (Hd= 0,590-0,833) and low nucleotide diversity (Pi= 0,05-0,65) values can be explained by the presence of small populations sensitive to genetic drift and founder effects. Phylogenetic analyses constructed with neihgbour joining, maximum likelihood and maximum parsimony generated similar topologies supported by high bootstrap values. Phylogenetic tree topologies showed that the C. apoensis haplotype was located in the C. luteus species. Therefore, the validity of species status of C. apoensis should be checked morphologically. On the other hand, since the Kahta population in C. luteus has a remarkably high genetic diversity, it must be re-evaluated morphologically. The tectonic uplift of the Anatolian Plateau between the African and European plates during the Pliocene period may have probably prevented the presence of Carasobarbus in the west of the Anatolian diagonal.

DNA BARCODING USING COI GENE SEQUENCES OF WILD BETTA FIGHTING FISH FROM INDONESIA: PHYLOGENY, STATUS AND DIVERSITY

The wild betta fish is a potential ornamental fish export commodity normally caught by traders or hobbyists in the wild. However, the population of wild betta in nature has declined and become a threat for their sustainability. This research was conducted to analyze the genetic diversity, phylogenetic relationships, and molecular identification through DNA COI gene sequence of Indonesian wild betta fish. A total of 92 wild betta fish specimens were collected in this study. Amplification of COI genes was carried out using Fish F1, Fish R1, Fish F2, and Fish R2 primers. The genetic diversity and phylogenetic relationships were analyzed using MEGA version 5 software program. Species identification of the specimen was conducted using BLAST program with 98-100% similarity value of the DNA sequences to indicate the same species. Phylogenetic tree construction showed seven monophyletic clades and showed that Betta smaragdina was the ancestral species of genus Betta in Indonesian waters. Genetic distance among species ranged from 0.02 to 0.30, whereas intra-species genetic distance ranged from 0 to 6.54.

DNA barcoding of 4 species of cheyletid mites based on COI and 18S rRNA gene sequences

To analyze the sequences of mitochondrial cytochrome C oxidase subunit I gene (COI) and 18S ribosomal RNA gene (18S rRNA), so as to identify the feasible DNA barcodes for 4 species of cheyletid mites and improve the DNA barcoding database for cheyletid mites. Cheyletid mite samples were collected from small-scale flour mills in Fuyang, Wuhu and Tongling cities of Anhui Province from May 2018 to July 2019, extracted and morphologically identified. Then, genomic DNA was extracted from a single cheyletid mite, and the COI and 18S rRNA gene sequences were obtained by PCR amplification, cloning and sequencing. The obtained sequences were aligned using the BLAST software. Multiple sequence alignment was done using the software ClustalX version 1.83 using the known gene sequences from cheyletid mites. The genetic distance was calculated using the software MEGA X, and the phylogenetic tree was created using the maximum likelihood method. The DNA barcoding results of Cheyletus malaccensis, C. carnifex and Cheletomorpha lepidopterorum were consistent with the morphological identification, while no sequences pertaining to Eucheyletia reticulate were retrieved in the GenBank database. The proportions of A + T were 69.6% and 55.1% in the COI and 18S rRNA sequences of 4 cheyletid mites species, respectively, and the numbers of base substitutions were 137 and 46, respectively. There were 154 to 321 and 58 to 99 inter-species variation loci in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, respectively, and the intra-species genetic distance was all 0.020 or less in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, with inter-species genetic distance of 0.235 to 0.583 and 0.078 to 0.114, respectively. Phylogenetic analysis based on COI and 18S rRNA genes showed that all four species of cheyletid mites were clustered into a branch with a 100% supportive rate, which was consistent with the morphological identification. Mitochondrial COI gene is superior to 18S rRNA gene as DNA barcodes for 4 species of cheyletid mites, which is more suitable to be used to investigate the phylogenetic relationship of at genus and species levels.

Genetic variation of native and introduced climbing perch Anabas testudineus (Bloch, 1792) derived from mitochondrial DNA analyses

The native and introduced Thai and Vietnamese Climbing perch Anabas testudineus are cultured in semi-intensive and intensive systems in Bangladesh. We sampled the native and the introduced A. testudineus from Bangladesh to test genetic variations based on Cytochrome C oxidase subunit I (COI) and Cytochrome (Cytb) gene. The COI nucleotide sequences of A. testudineus of India, Thailand, and Vietnam were retrieved from NCBI genebank and analyze. In the COI nucleotides, the higher AT content (53.6%) and lower GC content (46.4%) with larger differences were observed (values>0). The transition/transversion bias ( R ) based on Kimura 2-parameter (K2P) model was 8.44 with a higher transitional substitution (89%). The intraspecies genetic distance of COI nucleotide sequences based on a K2P model ranged from 0.000 to 0.094. The lowest genetic distance (0.00) was observed between the pair of reference and sequenced COI nucleotide sequences of Thai as well as Vietnamese A. testudineus , where very little genetic distance (0.002) was found between the sequenced Bangladeshi and reference Indian COI nucleotide sequences. The Thai and Vietnamese A. testudineus formed one cluster, whereas Bangladeshi and reference Indian A. testudineus formed a separate cluster in the phylogenetic tree using maximum likelihood method and time tree extrapolated from the molecular clock hypothesis. Our study provides evidence that the native and introduced A. testudineus are different lineages that might have evolved due to ecological variations at their origin. We suggest maintaining purity of breeding and cultured populations of the native and introduced A. testudineus for sustainable aquaculture in Bangladesh.

DNA barcoding elucidates the population genetic diversity of venomous cobra species (Reptilia: Elapidae) in Indo-Bangladesh region

The DNA barcode data of venomous cobra species (Naja naja and Naja kaouthia) are limited in the global database, especially from India and Bangladesh. Owing to the rapid success of DNA barcoding for discriminating a variety of species around the world, the present study aimed to generate the mitochondrial cytochrome c oxidase subunit I (COI) gene information of two morphologically identified deadly elapid species from the Mizoram state in northeast India and Rangpur in northern Bangladesh. The multiple species delimitation methods: Automatic Barcode Gap Discovery (ABGD), General Mixed Yule-Coalescent (GMYC), and Poisson-Tree-Processes (bPTP) revealed 14, 16, and 18 molecular operational taxonomic units (MOTUs) for 12 Naja species. The binocellate cobra, N. naja showed monophyletic clustering in both maximum-likelihood (ML) and Bayesian (BA) trees, single MOTU in ABGD and GMYC, and negligible intra-species genetic distance (0.2%) with two haplotypes. However, the monocellate cobra, N. kaouthia showed paraphyletic and polytomy in ML and BA phylogenies respectively; more than one MOTUs in ABGD, GMYC, and bPTP analyses; and sufficient intra-species genetic distances (0.6–2.3%) with five haplotypes related to the diverse geographical locations in Bangladesh, China, India, and Thailand. By superimposing the executed species delimitation criteria, the present molecular-based investigation concludes the presence of cryptic diversity of N. kaouthia in Indo-Bangladesh, China, and Thailand.

New Distributional Record of Oblique-barred Monocle Bream Scolopsis xenochrous, Günther, 1872 (Perciformes: Nemipteridae) from Wadge Bank of Southeastern Arabian Sea

The present study reports the Scolopsis xenochrous Gunther, 1872 off Vizhinjam coast, from Wadge Bank, Southeastern Arabian Sea. Four specimens of S. xenochrous were collected at a depth of 20 to 30 m by a gill net operated by traditional fishing craft. The morphometric and molecular identification characters of the verified specimens are described. The most of the morphometric measurement and meristic counts were confirmed with earlier explanation of S. xenochrous: Totally 45–46 lateral line scale, caudal fin deeply forked and upper lobe slightly lengthier than the lower lob and produced into a short or moderately long filament. Further, it has six oblique rows with thirty-two numbers of brown spots and an elongate white to bluish blotch below the lateral line. The present Indian specimen’s sequences were compared with other sequences of the S. xenochrous and other species of this genus obtained from the NCBI, BOLD, and Cryobank. The overall level of intra-species genetic distances was 2.7%, between Taiwan, Indonesia and Philippines were showed 0.0-0.5% of variation except for KY362992. The present study revealed the first record for the Wadge Bank coast of Southeastern Arabian Sea.

Species identification of zooplankton resting eggs based on DNA barcode technology: A case study of Lake Liuye (Changde), Lake Dongting Basin

以洞庭湖流域的典型城市湖泊常德柳叶湖表层沉积物中浮游动物休眠卵为研究对象，采用DNA条形码技术进行种类鉴定，从191个休眠卵中成功获得101条有效序列，鉴定成功率约为53%.根据NCBI数据库比对成功鉴定休眠卵9科12属11种，另有6个样品鉴定到属或科；3个类群的种间遗传距离平均为种内遗传距离的68倍，表明可以利用线粒体细胞色素c氧化酶第一亚基编码基因（COⅠ）对休眠卵进行有效物种鉴定.通过Neighbor-Joining树进行系统发育分析，发现所鉴定的物种与其参比序列聚类为一支，所有物种分别聚为独立的一个支系，不同物种可有效区分.研究结果均表明，COⅠ基因作为DNA条形码可以实现沉积物中浮游动物休眠卵的物种鉴定.;With the rapid development of modern molecular biology tools, especially DNA barcoding technology, a rapid, accurate and efficient identification method for species of zooplankton resting eggs is possible. In this study, zooplankton resting eggs, including copepods,cladocerans and rotifers in the surface sediments of Lake Liuye (Changde), a typical urban lake in Lake Dongting Basin, were studied. Using DNA barcode technology for species identification, 101 valid DNA sequences were successfully obtained from 191 resting eggs, and the identification success rate was 53%. According to the NCBI database comparison, 9 families, 12 genera and 11 species of resting eggs were successfully identified, and 6 samples were only identified to the genus or family level. The inter-species genetic distance of the three taxa was 68 times that of the intra-species genetic distance, indicating that the cytochrome oxidase Ⅰ (COⅠ) gene could be used for effective species identification of zooplankton resting eggs. Through the phylogenetic analysis by Neighbour-Joining tree, it was found that the identified species and their reference sequences were clustered into one branch, and all different species were clustered into independent branches respective. Therefore, different species could be effectively distinguished. The above results all indicate that the mitochondrial COⅠ gene, as a DNA barcode, can be used to identify the species of zooplankton resting eggs in sediment of lakes.

Phylogenetic tree analysis based on the 16S sequence alignment for Klebsiella spp. isolated from different sources

16S ribosomal RNA (16S rRNA) gene sequences used to study bacterial phylogeny and taxonomy have been by far the most common housekeeping genetic marker utilized for identification and ancestor determination. This study aimed to investigate, for the first time, the relationship between Klebsiella spp. isolated from clinical and environmental samples in Iraq.&#x0D; Fifty Klebsiella spp. isolates were isolated from clinical and environmental sources. Twenty-five isolates were collected from a fresh vegetable (Apium graveolens) and 25 from clinical samples (sputum, wound swab, urine). Enteric bacteria were isolated on selective and differential media and identified by an automatic identification system, vitek-2. The total DNA was extracted and PCR amplified for selected isolates. The 16S rRNA gene was amplified by using the universal primer 27F (5'- AGAGTTTGATCCTGGCTCAG- 3') and 1492R (5'- GGTTACCTTGTTACGACTT- 3’). The 16SrRNA gene sequence was analysed among some local isolates, and the results were compared with the standard data of similar registered strains in NCBI. The most common species of Klebsiella was Klebsiella pneumoniae pneumoniae (Kpp), followed by Klebsiella pneumoniae ozaenae (Kpo) and Klebsiella oxytoca (Ko). The results of the identification of species and sub species by using the biochemical test (vitek-2) were more precise than those obtained by the use of the universal primer.Phylogenetic tree strategies have clearly indicated a relatively close similarity amongst all analysed Klebsiella isolates and revealed the intra-species genetic distance between the individual isolates of the Klebsiella spp. In conclusion, our results revealed the main advantage of using universal primers for the identification of Klebsiella spp. and their root from nature.

Reassessment of species distribution and occurrence of mud crab (Scylla spp., Portunidae) in Malaysia through morphological and molecular identification.

This study utilized genetic and morphometric approaches to assess the molecular and morphometric differentiation among commercially important species of mud crab. Molecular investigations were derived from 542 bp mitochondrial DNA COI on 249 individuals within genus Scylla from nine states in Malaysia represents four marine regions; South China Sea, Sulu Sea, Straits of Singapore and Straits of Malacca. Four specimens were obtained from Indonesia to give a robust analysis in this study. For species delimitation, Automatic Barcode Gap Discovery (ABGD) method on a web interface was employed. Analysis on phylogenetics was implemented utilizing Neighbour joining (NJ) and Maximum Parsimony (MP) methods. The inter- and intraspecies genetic distances (Ds) was computed using Kimura 2-parameter distance and executed in MEGA version 5.05. All samples were genetically and morphologically identified and clustered into four distinct species. Among the species, S. olivacea was the most abundant (n = 111), on the other hand the occurrence of S. paramamosain in Malaysia was very low (n = 29). No single individual of S. serrata from Malaysia was recorded in this study. Both genetic distance and phylogenetic approaches exhibited a correlative monophyletic association among all specimens analysed. This present study is crucial as it reports the reassessment of all species within genus Scylla in Malaysia, eventually could be employed as a reference source for subsequent research mainly on mariculture and other conservation efforts for the species.