Kimura 2-parameter Research Articles

Genetic structure and phylogenetic relationships of Phyllidiellapustulosa species from Seribu Islands, North Sulawesi, Halmahera, and West Papua

Phyllidiella pustulosa are brightly coloured gastropod molluscs frequently found in coral reefs of the tropical Indo-Pacific. Phyllidiella pustulosa is widely distributed in Indonesia, such as Seribu Island, North Sulawesi, West Papua, and Halmahera. Based on the genetic characteristics of an individual’s DNA sequence, differences between species can be identified. In this paper, we would like to provide the molecular analysis and phylogenetic relationship among nudibranchs from Indonesian waters. Identification was made by measuring the genetic distance between species. The phylogenetic tree reconstruction was made using the Kimura 2-parameter model with 1000 times bootstrap with neighbor-joining and maximum likelihood method. There is 46 DNA Sequence obtained from 4 different regions (Seribu Island, Halmahera, North Sulawesi, and West Papua). The genetic distance of West Papua and Halmahera has the smallest value among other populations, which is between 0.0051-1.4629, compared to the population in Halmahera. The phylogenetic tree also shows populations from West Papua and Halmahera are on the same lineage, indicating that the population in West Papua and Halmahera had the closest relation. The study suggested that North Sulawesi, Halmahera and West Papua have genetic mixing of the same region, which is distinctive from Seribu Island.

DNA Barcoding of Mosquitoes from the Pantanos de Centla Biosphere Reserve, Southeastern Mexico.

Accurate identification of mosquito species is essential to support programs that involve the study of distribution and mosquito control. Numerous mosquito species are difficult to identify based only on morphological characteristics, due to the morphological similarities in different life stages and large numbers of some species that are members of morphologically similar species complexes. In the present study, the mosquitoes collected in the Pantanos de Centla Biosphere Reserve, southeastern Mexico, were evaluated using a combination of morphological and molecular approaches (mitochondrial cytochrome c oxidase subunit I [COI] DNA barcode). A total of 1,576 specimens of 10 genera and 35 species, mostly adult stages, were collected. A total of 225 COI DNA barcode sequences were analyzed; most species formed well-supported groups in the neighbor joining, maximum likelihood, and Bayesian inference trees. The intraspecific Kimura 2-parameter (K2P) genetic distance averaged 1.52%. An intraspecific K2P distance of 6.20% was observed in Anopheles crucians s.l., while a deep split was identified in Culex erraticus and Cx. conspirator. This study showed that COI DNA barcodes offer a reliable approach to support mosquito species identification in Mexico.

Genomic and biological characteristics of an alphabaculovirus isolated from Trabala vishnou gigantina

The oak lappet moth, Trabala vishnou gigantina is a forest insect pest that damages broad-leaf trees severely. Trabala vishnou gigantina nucleopolyhedrovirus (TrviNPV) has been isolated from a naturally infected T. vishnou gigantina larva and investigated for its biology and the potential to be a biological control agent against its insect host. TrviNPV was characterized by electron microscope of occlusion bodies (OBs), genomic sequencing and field control efficacy. TrviNPV OBs exhibited an irregular polyhedral shape varying in size from 0.99 to 3.99 μm with multiple nucleocapsids per virion. The genome of this virus was 165 657 bp in length with 40.33% GC content and encoded 146 putative ORFs including the 38 baculovirus core genes. TrviNPV is a group II alphabaculovirus that encodes F protein and lacks the gp64 gene specific to group I alphabaculoviruses. Phylogeny and Kimura-2 parameter analysis revealed TrviNPV to be a novel species and closest to ArdiNPV, EupsNPV and OrleNPV. Bioassays and field trials in a shrubland revealed that TrviNPV was virulent and effective to control T. vishnou gigantina in arid semi-desert region. This work firstly reported the whole genome of TrviNPV as well as its biological characters for a possibility to develop this virus as bio-pesticide in the future.

New species of Labiobaetis Novikova & Kluge from Southeast Asia and New Guinea (Ephemeroptera, Baetidae).

Material collected between 2006 and 2016 in Borneo, Sulawesi, and New Guinea further increased our knowledge of Labiobaetis Novikova & Kluge in these regions. Five species were previously reported from Borneo, two from Sulawesi, and 33 from New Guinea. Six new species have been identified using a combination of morphology and genetic distance (COI, Kimura 2-parameter), one species from Borneo (Brunei), one from Sulawesi, and four from New Guinea. They are described and illustrated based on their larvae and keys to the species of the relevant groups are provided. Additionally, new reports, a complementary description, and the COI sequence for L.dendrisetis Kaltenbach & Gattolliat are presented. The distribution of Labiobaetis in the Wallacea region is discussed based on the new findings. The total number of Labiobaetis species worldwide is augmented to 153.

Molecular identification and phylogenetic analysis of Papaver based on ITS2 barcoding.

In forensic cases suspected to involve Papaver somniferum, species identification is key to the investigation. To accurately detect and identify P.somniferum as well as common adulterants of the same genus, 19 internal transcribed spacer 2 (ITS2) sequences of P.somniferum (256bp), Papaver canescens (254bp), Papaver nudicaule (254bp), Papaver pavoninum (250bp), Papaver radicatum (254bp), and Papaver rhoeas (256bp) were obtained. Based on the ITS2 sequence, similarity analysis via BLAST, the nearest Kimura-2-parameter (K2P) genetic distances were calculated, and a phylogenetic tree was constructed using MEGA X software for the identification of six species of Papaver. Finally, differences in the ITS2 secondary structure between species were analyzed. The best matches of the P.somniferum ITS2 sequence were of other P.somniferum from different sources. The nearest K2P genetic distances between P.somniferum and its counterparts from other sources were zero, which was the smallest pairwise genetic distance among distances from the other five Papaver species. Various sources of P.somniferum clustered into an independent branch in the phylogenetic tree. The secondary structures of P.somniferum and P.rhoeas were significantly different from those of the other four species of Papaver. In summary, P.somniferum can be effectively distinguished from five closely related plants of the same genus by using ITS2 as a DNA barcode.

Mitochondrial phylogenetic and diversity analysis in Azi-Kheli buffalo.

The present study was conducted for the first time in Pakistan to investigate Cytochrome C Oxidase Subunit 1 (CO1) gene and full-length Displacement Loop (D-loop) region of mitochondrial DNA in Azi-Kheli buffalo breed native to northern hilly areas of Khyber Pakhtunkhwa Province of Pakistan. The present study was designed to investigate phylogeny and diversity in Azi-Kheli buffalo, through two mitochondrial DNA regions, i.e., Cytochrome C Oxidase Subunit-I (CO1) and Displacement Loop (D-loop) region. Thirty (30) blood samples were taken from Azi-Kheli pure breed animals from original breeding tract, i.e., Khwazakhela, Swat. Polymerase chain reactions using gene-specific primers were carried out for amplifying 709-bp region of CO1 gene and 1159-bp region of D-Loop for identification, phylogeny, and diversity in Azi-Kheli buffalo, respectively. The sequences of CO1 gene revealed four (04) haplotypes, whereas D-loop sequences revealed five (05) haplotypes. Mean interspecific diversity with related species was 2.56%, and mean intraspecific diversity within Azi-Kheli buffalo was 0.25%, estimated via Kimura-2 parameter. Phylogenetic tree (maximum likelihood) revealed clustering of Azi-Kheli haplotypes with river buffalo and is distinct from swamp buffalo and other related species of genus Bubalus. Mean haplotype and nucleotide diversity of D-loop were Hd = 0.9601 ± SD = 0.096 and π = 0.01208 ± SD = 0.00182, respectively. Phylogenetic tree (neighbor-joining) revealed two main clades, i.e., river buffalo and swamp buffalo clade. The haplotypes of Azi-Kheli clustered with haplotypes of different river buffalo breeds at different positions. The current study suggests that Azi-Kheli has common origin with other river buffalo breeds; hence, it is river buffalo which harbors high genetic diversity.

Use of rbcL and ITS2 for DNA barcoding and identification of Solanaceae plants in hilly state of Mizoram, India

Solanaceae is an important angiosperm family having high economic and commercial importance being used as sources of food, spice, and medicine. The traditional method of plant identification using morphological characters often leads to inaccurate and unreliable results because of genetic and environmental factors. In Solanaceae family, highly similar morphological traits among the species also make the identification difficult. The present study evaluated the viability of employing DNA barcodes, ITS2 and rbcL regions to identify Solanaceae species. DNA barcoding is a rapid and reliable tool to identify and study the genetic relationship among plants and animals. The present study for the first time was conducted during 2017 to 2019 at the Department of Botany, Mizoram University to analyze the applicability of two DNA regions-rbcL (Ribulosebisphosphate Carboxylases) and ITS2 (Internal Transcribed Spacer gene), as the barcodes for the identification of Solanaceae species of Mizoram, India. Fifteen Solanaceae species were analyzed at ITS2 and rbcL regions. The success rates of PCR amplification and sequencing of ITS2 and rbcL were 100% and MEGA 7.0 was used to align the sequences and to compute genetic distances. The results from BLAST and nearest-distance methods revealed that ITS2 and rbcL can be used to identify all the studied species and high divergences at taxonomic levels were also found using K2P (Kimura 2-parameter) model. Hence, our study reveals that rbcL and ITS2 regions are suitable DNA barcodes for the identification of Solanaceae species and therefore can be successfully used to monitor the adulteration of plants.

Updated distribution of anopheline mosquitoes in Hokkaido, Japan, and the first evidence of Anopheles belenrae in Japan

BackgroundIn Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015.MethodsA survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2.ResultsA total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido.ConclusionThe updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.Graphical abstract

Molecular characterization and genetic diversity of four undescribed novel oleaginous Mortierella alpina strains from Libya

Background: A large number of undiscovered fungal species still exist on earth, which can be useful for bioprospecting, particularly for single cell oil (SCO) production. Mortierella is one of the significant genera in this field and contains about hundred species. Moreover, M. alpina is the main single cell oil producer at commercial scale under this genus. Methods: Soil samples from four unique locations of North-East Libya were collected for the isolation of oleaginous Mortierella alpina strains by a serial dilution method. Morphological identification was carried out using light microscopy (Olympus, Japan) and genetic diversity of the isolated Mortierella alpina strains was assessed using conserved internal transcribed spacer (ITS) gene sequences available on the NCBI GenBank database for the confirmation of novelty. The nucleotide sequences reported in this study have been deposited at GenBank (accession no. MZ298831:MZ298835). The MultAlin program was used to align the sequences of closely related strains. The DNA sequences were analyzed for phylogenetic relationships by molecular evolutionary genetic analysis using MEGA X software consisting of Clustal_X v.2.1 for multiple sequence alignment. The neighbour-joining tree was constructed using the Kimura 2-parameter substitution model. Results: The present research study confirms four oleaginous fungal isolates from Libyan soil. These isolates (barcoded as MSU-101, MSU-201, MSU-401 and MSU-501) were discovered and reported for the first time from diverse soil samples of district Aljabal Al-Akhdar in North-East Libya and fall in the class: Zygomycetes; order: Mortierellales. Conclusions: Four oleaginous fungal isolates barcoded as MSU-101, MSU-201, MSU-401 and MSU-501 were identified and confirmed by morphological and molecular analysis. These fungal isolates showed highest similarity with Mortierella alpina species and can be potentialistic single cell oil producers. Thus, the present research study provides insight to the unseen fungal diversity and contributes to more comprehensive Mortierella alpina reference collections worldwide.

Molecular characterization and genetic diversity of four undescribed novel oleaginous Mortierella alpina strains from Libya.

Background: A large number of undiscovered fungal species still exist on earth, which can be useful for bioprospecting, particularly for single cell oil (SCO) production. Mortierella is one of the significant genera in this field and contains about hundred species. Moreover, M. alpina is the main single cell oil producer at commercial scale under this genus. Methods: Soil samples from four unique locations of North-East Libya were collected for the isolation of oleaginous Mortierellaalpina strains by a serial dilution method. Morphological identification was carried out using light microscopy (Olympus, Japan) and genetic diversity of the isolated Mortierella alpina strains was assessed using conserved internal transcribed spacer (ITS) gene sequences available on the NCBI GenBank database for the confirmation of novelty. The nucleotide sequences reported in this study have been deposited at GenBank (accession no. MZ298831:MZ298835). The MultAlin program was used to align the sequences of closely related strains. The DNA sequences were analyzed for phylogenetic relationships by molecular evolutionary genetic analysis using MEGA X software consisting of Clustal_X v.2.1 for multiple sequence alignment. The neighbour-joining tree was constructed using the Kimura 2-parameter substitution model. Results: The present research study confirms four oleaginous fungal isolates from Libyan soil. These isolates (barcoded as MSU-101, MSU-201, MSU-401 and MSU-501) were discovered and reported for the first time from diverse soil samples of district Aljabal Al-Akhdar in North-East Libya and fall in the class: Zygomycetes; order: Mortierellales. Conclusions: Four oleaginous fungal isolates barcoded as MSU-101, MSU-201, MSU-401 and MSU-501 were identified and confirmed by morphological and molecular analysis. These fungal isolates showed highest similarity with Mortierella alpina species and can be potentialistic single cell oil producers. Thus, the present research study provides insight to the unseen fungal diversity and contributes to more comprehensive Mortierella alpina reference collections worldwide.

Distinguishing level-1 phylogenetic networks on the basis of data generated by Markov processes

Phylogenetic networks can represent evolutionary events that cannot be described by phylogenetic trees. These networks are able to incorporate reticulate evolutionary events such as hybridization, introgression, and lateral gene transfer. Recently, network-based Markov models of DNA sequence evolution have been introduced along with model-based methods for reconstructing phylogenetic networks. For these methods to be consistent, the network parameter needs to be identifiable from data generated under the model. Here, we show that the semi-directed network parameter of a triangle-free, level-1 network model with any fixed number of reticulation vertices is generically identifiable under the Jukes–Cantor, Kimura 2-parameter, or Kimura 3-parameter constraints.

First Report of Golovinomyces orontii Causing Hydrangea macrophylla Powdery Mildew in Qinghai-Tibet Plateau, China.

Hydrangea macrophylla (Thunb.) Ser. (Hydrangeaceae) is the most popular hydrangea species grown in home gardens and landscapes in China. Plants of H. macrophylla with symptoms of powdery mildew were found in a commercial wholesale nursery in Huzhu, Haidong (36°49'11.87" N, 101°57'03.36″E, alt. 2490 m), in May 2020, with disease incidence reaching 80%. Symptoms included yellowing and necrosis of leaves. Upon microscopic observation, masses of conidia and mycelium were observed covering the symptomatic tissues. Fungal isolates displayed nipple-shaped hyphal appressoria, often poorly developed, conidiophores erect, arising laterally or from the upper surface of hyphal mother cells, and positioned almost centrally or towards one end of the cells, up to about 160 μm long (n = 30), with foot cells straight or flexuous, 32 to 86 × 8 to 13 μm (n = 50), followed by one to three shorter cells about 11 to 24 × 10 to 15 μm (n = 50), forming catenescent conidia in usually predominantly chains, conidia doliiform to limoniform, hyaline, 24 to 35 × 13 to 25μm (n = 50). Conidial germination was of the Euoidium type. Chasmothecia were not observed. To confirm fungal classification, single spores were isolated and cultured on detached leaf bioassay following the protocol described in Farinas et al. (2019). Total DNA was extracted directly from single-spore cultures using a Chelex extraction method (Walsh et al. 1991). The rDNA internal transcribed spacer (ITS) regions were amplified by polymerase chain reaction (PCR) utilizing the universal primer pairs ITS1/ITS4 (White et al. 1990). The sequences (726-727 bp) were deposited in GenBank (accessions no. MT568633, MT757924 and MT757925). The ITS sequences showed 99.9-100% identity with a sequence of Golovinomyces orontii reported on Papaver rhoeas (AB769466) in Switzerland. Based on the ITS rDNA phylogenetic tree, the sequences retrieved from the specimen clustered within a strongly supported clade with G. orontii (AB769466), confirming the identity of the pathogen (Takamatsu 2013). Cladistic trees were constructed using the neighbor-joining method with the Kimura 2-parameter substitution model in MEGA 6.0. Branch robustness was assessed via bootstrap analysis with 1,000 replicates. To confirm pathogenicity, eight H. macrophylla plants were sprayed until run-off with a suspension containing 1 × 105 conidia/ml. Four plants were used for fulfilling Koch's postulates and four plants were used as mock-inoculated controls sprayed only with sterile distilled water. Inoculated and non-inoculated plants were covered with plastic bags separately and maintained overnight in a greenhouse at 25 ± 2°C and 50 to 60% relative humidity. Typical powdery mildew colonies developed on inoculated plants 10 to 15 days after inoculation, which were morphologically identical to those originally observed on the diseased plats, whereas the control plants remained symptomless. To our knowledge, this is the first report of powdery mildew caused by G. orontii on H. macrophylla in Qinghai-Tibet Plateau, China (Braun and Cook 2012).

DNA barcoding of the mesic adapted striped mouse, Rhabdomys dilectus in the Eastern Cape and KwaZulu-Natal provinces of South Africa

AbstractSouth African small mammals are under-represented in DNA barcoding efforts, particularly from the eastern forested regions of the country. This study reports DNA barcoding ofRhabdomystaxa from previously unsampled parts of the Eastern Cape and KwaZulu-Natal provinces of South Africa. The complete mitochondrial DNA cytochrome oxidase I (COI) gene was sequenced for 101Rhabdomyssp. individuals from 16 localities from all three main forest groups (coastal, mistbelt, and scarp forests). Molecular data were supplemented with external morphological measurements, including those deemed potential taxonomically diagnostic characters. Findings indicate the area to be inhabited solely byRhabdomys dilectus chakae. Haplotypes distributed across the three forest groups were separated by shallow sequence divergences ranging from 0.001–0.015 (Kimura 2-parameter model) and displayed very little population genetic structure (FST= 0.071787). Morphological data revealed some regional metric differences in external morphology, but all the head-and-body to tail (HB: tail) ratios match that ofR. d. chakae, and consequently, molecular and morphological data are congruent. These data confirm a range extension ofR. d. chakae, supporting the utility of COI barcodes in the identification of small mammalian species.

DNA barcodes provide evidence for the independent origin of the cultivated silkwormsSamia ricini andSamia cynthia

Samia ricini (Wm. Jones) andSamia cynthia (Drury) (Lepidoptera: Saturniidae) have been used as traditional sources of food as well as silk-producing insects. However, the phylogenetic relationship between the two silkworms remains to be addressed. In this study, the mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences corresponding to DNA barcodes from 13Samia species were analysed, and a DNA barcode-based phylogenetic framework for theseSamia species was provided. Phylogenetic analysis showed that multiple individuals of a species could be clustered together. Our analysis revealed a close relationship amongSamia yayukae Paukstadt, Peigler and Paukstadt,Samia abrerai Naumann and Peigler,Samia kohlli Naumann and Peigler,Samia naessigi Naumann and Peigler,Samia naumanni Paukstadt, Peigler and Paukstadt, andSamia kalimantanensis Paukstadt and Paukstadt. The mixed clustering relationship and low Kimura-2-parameter (K2P) genetic distance (0.006) between individuals ofS. ricini andSamia canningi (Hutton) indicated that the cultivated silkwormS. ricini was derived from the non-cultivated silkwormS. canningi. The remote phylogenetic relationship and high K2P genetic distance (0.039) indicated thatS. ricini andS. cynthia are distinct species, thus providing solid molecular evidence that they had entirely independent origins. The relationships betweenS. kalimantanensis andS. naumanni and betweenS. cynthia andSamia wangi Naumann and Peigler, as well as the potential cryptic species withinS. abrerai were also discussed. This is the first study to assess the DNA barcodes of the genusSamia, which supplements the knowledge of species identification and provides the first molecular phylogenetic framework forSamia species.

شناسایی مولکولی ماهیان زینتی بومی آفانیوس زاگرس (Aphanius vladykovi) و آفانیوس گنو (Aphanius ginaonis) با استفاده از ژن سیتوکرم اکسیداز در ایران

This research was carried out with the aim of molecular identification and using cytochrome oxidase gene of two species of ornamental fish native to Iran entitled Aphanius vladykovi and Aphanius ginaonis. Five samples of each fish were collected from Sarcheshmeh Park (Aloni city, ­Chaharmahal and Bakhtiari province) and Genu spring (Bandar Abbas) and after morphological identification and DNA extraction, they were sequenced. Then using BioEdit software and the NCBI database, the amount of homology of the obtained sequences was measured. The analysis of the evolution tree was performed by the closest proximity and parsimony method based on the Kimura 2-parameter model using mega software. The results showed that these species were genetically close to each other in different parts of Fars province (A. vladykovi) and Turkey, and a clade with two sister clusters next to the species belonging to the northern offerings more than Turkey includes Aphanius anatoliae, Aphanius sureyanus, Aphanius maeandricus and Aphanius iconii. The greatest genetic differences can be observed between the specimens of Aphanius vladykovi from Sarcheshmeh Park spring and Aphanius sureyanus from Turkey, which was not unexpected due to the deep geographical gap between the mentioned regions. At the same time, the species Aphanius Genu shows itself in a separate clade with a large genetic distance.­ Various geological factors caused Genu fish to separate itself from other studied species and to hide in Hormozgan region. As a result, ­the gene flow between this region and other regions has stopped, and caused the Aphanius ginaonis in Hormozgan province to remain as a unique species.

First Report on the Characterization of Genetic Diversity of Philippine-reared Bombyx mori Strains Based on COI and ITS2

The mulberry silkworm, Bombyx mori, is known to produce silk and was introduced to the Philippines from China, Japan, Korea, and India. The silkworm germplasm in the Philippines is sourced from three centers located in regions that vary in climatic conditions: Philippine Textile Research Institute–Technology Center Misamis Oriental (PTRI-TCMO); Department of Science and Technology– Cordillera Administrative Region (CAR) in La Trinidad, Benguet; and the Don Mariano Marcos Memorial State University–Sericulture Research and Development Institute (DMMMSU-SRDI), La Union. To date, associated information with the centers’ germplasm constitutes differences in biological and productive traits such as yield and quality of raw silk. It lacks genetic information on strain diversity, which provide basis for the efficient selection of parental strains with traits of economic interest. This study aimed to characterize, for the first time, the genetic diversity of 17 pure lines as parental strains. Genetic variation was evaluated using the cytochrome c oxidase 1 (COI) gene and internal transcribed spacer 2 (ITS2). BLAST analysis of the COI and ITS2 sequences confirmed the identity of B. mori strains. Phylogenetic trees constructed from COI sequences formed a monophyletic clade, while the trees based on ITS2 sequences formed two distinct clusters. The pairwise distances based on the Kimura 2-parameter (K2P) model between the B. moristrains ranged from 0–0.003 for COI and from 0–0.002 for ITS2, indicating low genetic variation for the corresponding molecular markers. Variation in the COI gene determined four nucleotide changes while variations in the ITS2 sequences determined four to six nucleotide changes, except for one strain with higher nucleotide change. This study reports that the use of COI and ITS2 as markers was not able to genotypically differentiate the 17 strains, which appear different in terms of phenotypic characters, suggesting the use of other molecular markers to characterize not only the 17 strains but all other strains in the germplasm.

Identification of Cannabis Sativa L. Based on rbcL Sequence.

Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation.

A new species and first record of the genus Procerobaetis Kaltenbach & Gattolliat, 2020 (Ephemeroptera, Baetidae) from Thailand.

The genus Procerobaetis Kaltenbach & Gattolliat, 2020 is reported for the first time from Thailand, and Procerobaetis totuspinosussp. nov. is described as a new species based on larvae. It can be easily distinguished from other known Procerobaetis species by the presence of triangular spines at the posterior margin of tergites VI–IX. COI sequences were obtained from all known species. The genetic distances (Kimura 2-parameter) between the new species and the other species are between 20% and 23%. The morphological characters of the new species and its closely related species are discussed; larval key to all species of the genus Procerobaetis is also provided.

Application of COI gene for identification of some economically and forensically important muscid flies of India (Diptera: Muscidae)

Muscid flies are of great medical and veterinary importance because of their ability to transmit diseases to humans and animals. Their larvae are found on dead bodies and are used as a forensic indicator for estimating Post-Mortem Interval (PMI). A drawback of morphological identification of these larvae is lack of an identification key which covers all the life stages of the taxa. To overcome the problem, entomologists now focus on the alternative methods, which can speed up the identification process. Barcoding region of Cytochrome oxidase I (COI)l is believed to be the best among other markers for precise identification and to mark out phylogeny. During the present study, total 63 specimens of 24 species belonging to 7 genera representing the various muscid subfamilies were used to check the efficacy of COI gene in identification. 11 species were collected from various regions (Punjab, Haryana, Himachal Pradesh, Uttarakhand) of North India during the years 2015–2019, and sequences of rest of the species were downloaded from the database of Genbank. The percentage of intraspecific divergences ranged from 0%-0.5% for species of India and 0%-3.0% for the sequences from other countries. The interspecific divergence ranged from 3.1% to 17.3% and 3.3% to 16.7% respectively for Indian species and species from the other part of the world. 1000 bootstrapped Neighbor-joining with Kimura-2 parameter phylogenetic tree constructed based on COI gene sequences, clearly separated 24 species of these Muscid flies. This is the first ever study on COI gene based identification of medico-legal, veterinary and forensically important Muscid flies from India.

DNA barcode of Enggano hill myna, Gracula religiosa enganensis (Aves: Sturnidae) based on mitochondrial DNA cytochrome oxidase subunit I

Abstract. Jarulis, Muslim C, Kamilah SN, Utama AF, Permana D, Sari MM, Prayitno AH, Jannah IM. 2021. DNA barcode of Enggano hill myna, Gracula religiosa enganensis (Aves: Sturnidae) based on mitochondrial DNA cytochrome oxidase subunit I. Biodiversitas 22: 1635-1643. The sharp decline of the Enggano hill myna population due to illegal trading and habitat degradation needs to be our concern to prevent this bird from extinction. Taxonomically, Enggano hill myna is referred to as a sub-species, but this has not been confirmed by genetic data. We have sequenced seventeen Enggano hill myna mitochondrial DNA COI genes to describe their genetic identity (barcode), genetic distances, and phylogeny. DNA genome from seventeen blood samples was isolated with DNeasy® Blood and Tissue Kit Qiagen, while PCR amplification was performed using a pair primers, namely COIGRF (5'-TTCTGATTCTTTGGCCATCC-3') and COIGRR (5'-GTTGGAAGGCTTTGCGTTTA-3'). We used Clustal W alignment in MEGA 10.2.2 software to search single nucleotide polymorphisms. Genetic distance was analyzed by using the Kimura 2-parameter, and the phylogenetic tree was reconstructed with Neighbor-Joining models. We found 98.60% conservative sites, 0.69% parsimony sites, and 0.83% singleton sites from the 716 bp sequence. The highest nucleotide composition was cytosine (32.20%), and the lowest was guanine (16.80%), followed by 49% GC content. Seven SNP sites were found in 716 bp COI gene sequences of seventeen individuals. The genetic distance between Enggano hill myna individuals was ranged from 0.0-0.8%, and all Enggano hill myna individuals separated from Chinese and Indian populations in the phylogenetic tree with a genetic distance of 0.9% and 1.1%. Our data suggest that the Enggano hill myna population is still classified as a sub-species. The COI gene sequences that we found can be used to quickly identify this species and are also important to prevent illegal trading in Indonesia.