Kimura 2-parameter Research Articles

The Use of DNA Barcoding and Phylogenetic Analysis to Improve Identification of <i>Usnea</i> spp. Based on ITS rDNA

Lichen of the genus Usnea is quite common being used as a traditional herbal remedy. This genus is characterized by thallus, which is very similar among the species, leads to some difficulties in distinguishing them. In Indonesia, such research report on the availability of this genus based on their morphological characteristic is minimal. This might be due to too high morphological similarities among them. The molecular character, which is based on the DNA Barcode of Internal Transcribed Spacer (ITS) rDNA sequences, with its conserved region (5.8S) and varied region (ITS1 and ITS2), are becoming essential characters on identifying as well as analyzing the phylogenetic. The current study then proposed to identify and draw the species dendrogram of species within the Usnea genus obtained from Mount Lawu Forest of Central Java and Turgo Forest of Yogyakarta based on their phylogenetic and phenetic analysis. The dendrogram was constructed with UPGMA using the simple matching coefficient, whereas the phylogenetic tree was constructed with Maximum Likelihood (ML) using Kimura-2 parameter with 1000 bootstrap. The data were unable to draw phenetic relationships among the subgenus Usnea and Eumitria members. The phylogenetic tree shows the primary two clades, distinguishing the subgenus Usnea and Eumitria. The ITS rDNA sequence was able to identify most of the Usnea species.

A multilocus assessment reveals two new synonymies for East Asian Cyclommatus stag beetles (Coleoptera, Lucanidae).

Cyclommatus scutellaris Möllenkamp, 1912, Cyclommatus elsae Kriesche, 1921 and Cyclommatus tamdaoensis Fujita, 2010 are East Asian stag beetle species with long-debated taxonomic relationships due to high intraspecific morphological variability. In this study, we applied multilocus phylogenetic analyses to reassess their relationships. Two mitochondrial genes (16S rDNA, COI) and two nuclear genes (28S rDNA, Wingless) were used to reconstruct the phylogeny through the Bayesian inference (BI) and Maximum Likelihood (ML) methods. Both topologies supported two clades: the clade C. scutellaris was sister to the clade (C. elsae + C. tamdaoensis) with the subclade C. tamdaoensis embedded in the subclade C. elsae. The Kimura 2-parameter (K2P) genetic distance analysis yielded a low mean value (≤0.035) among the three taxa, which was well below the minimum mean value between other Cyclommatus species (≥0.122). We also compared the accuracy and efficiency of two approaches, GMYC and ABGD, in delimitating the three lineages. The result shows that ABGD is a better approach than GMYC. Our molecular data recognizes the three species as different populations of a single species, ranging from Taiwan Island to the continent. Therefore, we propose two new junior synonyms for C. scutellaris: C. tamdaoensis, syn. nov. and C. elsaesyn. nov.

Labiobaetis Novikova & Kluge in Ethiopia (Ephemeroptera, Baetidae), with description of a new species

Material collected between 2017 and 2019 in Ethiopia in the Awash River catchment substantially increased our knowledge of Labiobaetis Novikova & Kluge in this country. Four species were previously reported based on ecological investigations of Ethiopian rivers: L. glaucus (Agnew, 1961), L. latus (Agnew, 1961), L. vinosus (Barnard, 1932) and L. bellus (Barnard, 1932). We have identified six different species using a combination of morphology and genetic distance (COI, Kimura 2-parameter). Two of them, L. alahmadii Gattolliat & Al Dhafer, 2018 and L. potamoticus Gattolliat & Al Dhafer, 2018 were previously assumed to be endemic to the Arabian Peninsula. The status of L. bellus is discussed and remains unresolved. One species is new to science; it is described and illustrated based on its nymphs. A key to the nymphs of all Ethiopian species is provided. The interspecific K2P distances in Ethiopia are between 17% and 23%, the intraspecific distances are usually between 0% and 1%. The total number of Labiobaetis species worldwide is augmented to 145. The Afrotropical species of Labiobaetis are discussed in comparison to the species of other realms.

Genetic Variation Analysis of Four Local Varieties of Indonesian Black Rice (Oryza sativa L.) Based on Partially rbcL cpDNA Gene Sequence

Black rice ( Oryza sativa L.) varieties i.e. Toraja (South Sulawesi), Cempo Ireng (Yogyakarta), Wojalaka (East Nusa Tenggara), and Manggarai (East Nusa Tenggara) are four local black rice varieties in Indonesia whose character has not been widely studied, especially the character of genetic variation. Research aimed to determine the variation of the rbc L gene in the four local black rice varieties. The sample for testing the variation of the rbc L gene sequence in the form of black rice leaves six weeks after planting. Dendogram was carried out using the UPGMA method with the Kimura 2-parameter algorithmic calculation model using the MEGA5 version 5.2.2 program. The results showed that partially the rbc L gene sequence was successfully amplified on four black rice varieties with a sequence length of 487 bp. The partial rbc L sequence of black rice consisted of 26.58% tyrosine, 21.38% cytosine, 28.86% adenine, and 23.18% guanine. The value of G + C content was 0.446, with the frequency of invariable sites of 97.13%. The frequency of informative parsimony sites was 1.43% with a nucleotide diversity (Pi) value of 42-10, the number of haplotypes was 5, and the total number of mutations and polymorphic sites was 14. The ratio between transition and transversion (ts/tv ratio k) for purine bases was 1.741 and pyrimidine was 3.571, with the estimated overall ratio between transition and transversion (R) of 1.31. Based on the dendogram, the farthest genetic distance was found in Wojalaka and Manggarai varieties, which were 0.019 respectively. Keywords: black rice, genetic variation, local varieties, rbc L gen

New color patterns and new synonym of Odontolabis sinensis (Westwood, 1848) (Coleoptera: Lucanidae): insights from a multilocus phylogeny and species delimitation.

Taxonomy of Odontolabis sinensis (Westwood, 1848) and Odontolabis fallaciosa Boileau, 1901 has long been a controversial issue due to their highly morphological similarity except for elytral color patterns. In the present work, we assessed their taxonomic status by combining molecular phylogeny and species delimitation based on multilocus datasets (COI, 16S rDNA, Cytb, CAD, Wingless). Phylogenetic analyses under both Maximum Likelihood inference and Bayesian inference recovered identical topologies. All the examined individuals in the clade of O. sinensis and O. fallaciosa formed into five subclades belonging to four color patterns, respectively. The Kimura-2-Parameter (K2P) genetic distance analysis yielded a low mean value (0.0108-0.0295). The multiple species delimitations (ABGD, PTP and GMYC) reinforced them to be one MOTU. Our results suggest that these clades belong to the same species, and thus we propose O. fallaciosa to be a junior subjective synonym of O. sinensis. We also found two new color patterns corresponding to two clades from Southwestern China (north of 20°N and east of 105°E), respectively displaying the reddish-orange, slenderly rimmed patch and brownish-orange broad patches along the elytral edge, which was probably attributable to both historical and ecological factors in this particular region.

Genetic Relationship Between Hard Ticks (Ixodidae) Infesting Cattle from Select Areas of a Wildlife-Livestock Interface Ecosystem at Mikumi National Park, Tanzania.

Background: There has recently been a substantial increase in the number of tick species and tick-borne infectious agents in Tanzania. Owing to their impact on human, livestock, and wild animal health, increased knowledge of ticks is needed. So far, no published data on the genetic relationship between hard tick (Ixodidae) sequences collected from cattle are available in Tanzania. Methods: Ticks from cattle in the wards, which lie at the border of Mikumi National Park, were collected in the dry season, November to December 2019. Morphological identification of ticks was initially performed at the genus level. To identify ticks at the species level, molecular analysis based on the 16S rRNA gene was performed. Evolutionary relationships and genetic distances between ticks were determined using MaximumLikelihood and Kimura 2-parameter methods, respectively. Results: Based on morphology, two genera (Rhipicephalus and Hyalomma) were identified in the 630 adult ticks collected from a total of 252 cattle. Six species (Rhipicephalus microplus, Rhipicephalus evertsi, Hyalomma marginatum, Hyalomma rufipes, Hyalomma truncatum, and Hyalomma turanicum) were confirmed by BLASTn and phylogenetic analyses. Considerable mean and pairwise genetic distances were observed for Rhipicephalus and Hyalomma genera. Conclusion: The presence of different phylogenetic clusters and considerable mean and pairwise genetic distances observed reflect possible biological diversity of hard ticks present in the study area. Considering the value of the cattle in the livelihoods and economies of people and the country, the outcomes of this study will be useful in planning integrated control strategies for ticks and tick-borne diseases in Tanzania.

Mitochondrial genomes of two eucotylids as the first representatives from the superfamily Microphalloidea (Trematoda) and phylogenetic implications

BackgroundThe Eucotylidae Cohn, 1904 (Superfamily: Microphalloidea), is a family of digeneans parasitic in kidneys of birds as adults. The group is characterized by the high level of morphological similarities among genera and unclear systematic value of morphological characters traditionally used for their differentiation. In the present study, we sequenced the complete or nearly complete mitogenomes (mt genome) of two eucotylids representing the genera Tamerlania (T. zarudnyi) and Tanaisia (Tanaisia sp.). They represent the first sequenced mt genomes of any member of the superfamily Microphalloidea.MethodsA comparative mitogenomic analysis of the two newly sequenced eucotylids was conducted for the investigation of mitochondrial gene arrangement, contents and genetic distance. Phylogenetic position of the family Eucotylidae within the order Plagiorchiida was examined using nucleotide sequences of mitochondrial protein-coding genes (PCGs) plus RNAs using maximum likelihood (ML) and Bayesian inference (BI) methods. BI phylogeny based on concatenated amino acids sequences of PCGs was also conducted to determine possible effects of silent mutations.ResultsThe complete mt genome of T. zarudnyi was 16,188 bp and the nearly complete mt genome of Tanaisia sp. was 13,953 bp in length. A long string of additional amino acids (about 123 aa) at the 5′ end of the cox1 gene in both studied eucotylid mt genomes has resulted in the cox1 gene of eucotylids being longer than in all previously sequenced digeneans. The rrnL gene was also longer than previously reported in any digenean mitogenome sequenced so far. The TΨC and DHU loops of the tRNAs varied greatly between the two eucotylids while the anticodon loop was highly conserved. Phylogenetic analyses based on mtDNA nucleotide and amino acids sequences (as a separate set) positioned eucotylids as a sister group to all remaining members of the order Plagiorchiida. Both ML and BI phylogenies revealed the paraphyletic nature of the superfamily Gorgoderoidea and the suborder Xiphidiata.ConclusionsThe average sequence identity, combined nucleotide diversity and Kimura-2 parameter distances between the two eucotylid mitogenomes demonstrated that atp6, nad5, nad4L and nad6 genes are better markers than the traditionally used cox1 or nad1 for the species differentiation and population-level studies of eucotylids because of their higher variability. The position of the Dicrocoeliidae and Eucotylidae outside the clade uniting other xiphidiatan trematodes strengthened the argument for the need for re-evaluation of the taxonomic content of the Xiphidiata.Graphical

Morphology and phylogeny of scalopine moles (Eulipotyphla: Talpidae: Scalopini) from the eastern Himalayas, with descriptions of a new genus and species

AbstractAll scalopine moles are found in North America, except the Gansu mole (Scapanulus oweni), which is endemic to central-west China. In 2019, we collected two specimens of Scalopini on Mt Namjagbarwa in the eastern Himalayas, Tibet, China. We sequenced two mitochondrial (CYT B and 12S) and three nuclear (APOB, BRCA1 and RAG2) genes to estimate the phylogenetic relationships of the two moles, and also compared their morphology with other genera and species within the Scalopini. Both morphological and molecular analyses strongly suggest that the specimens represent a new monotypic genus and species, which are formally described here as Alpiscaptulus medogensis gen. et sp. nov. The dental formula of the new mole (44 teeth) is distinct from the Chinese Scapanulus oweni (36 teeth) and its hairy and pale brown tail is unique among species of the Scalopini. The Kimura-2-parameter (K2P) distances of CYT B between A. medogensis and the four recognized Scalopini genera range from 14.5% to 18.9%. A sister relationship between A. medogensis and Scapanulus oweni was strongly supported in the phylogenetic trees. The divergence between A. medogensis and Scapanulus oweni occurred in the mid-Miocene (c. 11.56 Mya), which corresponds with the rapid uplift of the Himalayan-Tibetan Plateau.

Philibaetis gen. nov., a new genus from the Philippines (Ephemeroptera, Baetidae)

Investigations of type material and new material from the Philippines (Luzon) revealed that Baetis luzonensis Müller-Liebenau, 1982 and B. realonae Müller-Liebenau, 1982 do not belong to Baetis Leach, 1815. A new genus, Philibaetisgen. nov., is described to accommodate both species and both are re-described based on larvae. The new genus is characterised by having a rectangular labrum with a submarginal row of long, simple setae on the dorsal surface and ventrally on lateral margins long, simple, spine-like setae, on anterolateral margins long, feathered setae and medially long, bifid setae and a partial, submarginal row of lanceolate setae. Both mandibles have blade-like incisors and dorsally, a mediolateral patch of long, spine-like setae; additionally, the left mandible has a tuft of long, partly branched setae at the base of the subtriangular process. Philibaetisgen. nov. is further characterised by a hypopharynx with a medial tuft of stout setae and anterolaterally, two smaller tufts of stout setae, a galea-lacinia with the distal denti-seta tooth-like and directed against canines, a fore femur apically with stout setae, both on anterior and posterior side and without a femoral patch and a claw with one row of denticles and two or three subapical setae. The protogonostyli under the cuticle of male last instar larvae are folded in the Labiobaetis type, excluding their affiliation to the genus Baetis. COI sequences were obtained from both species. The genetic distance (Kimura 2-parameter) between them is 17.5% on average. Very limited genetic distances of 0% to 3% (0.75% on average) were found between specimens of P. luzonensiscomb. nov.

DNA Mini-Barcodes, a Potential Weapon for Conservation and Combating Illegal Trade of Pangolin

Background Smuggling and illegal trade of pangolins and their scales has drastically reduced the wild population of pangolins. Accurate species identification is currently in urgent need as a powerful weapon for combating pangolin smuggling and trade and conserving the already endangered pangolin species. Aim of the study To develop an efficient method based on DNA mini-barcodes for accurate pangolin species identification and authentication of processed pangolin scales against the non-target species. Materials and methods The primers for amplifying the DNA mini-barcodes were designed based on cytochrome C oxidase subunit I (COI) gene fragments. The mini-barcodes were compared with the two universal barcodes (COI and Cytb) for performance in pangolin species identification by calculating the Kimura-2-parameter (K2P) distance, assessing the clustering dendrogram, and analyzing the BLAST similarity and barcoding gap. The accuracy of the three barcodes was also compared for authentication of pangolin scales against non-target species. Results Comparison of the three barcodes showed that the mini-barcode form COI had the highest amplification success rate (100%) and high variable sites (40.0%), with the ratio of mean inter- to intraspecific distance ratio was 25 and a distinct DNA barcoding gap. In the neighbor-joining (NJ) tree constructed based on the mini-barcode regions, each species of the pangolin family formed an obvious clade respectively, and the clades were all separated from those of the non-target species, indicating that the genetic information in the mini-barcode was sufficient for species identification. Conclusion The DNA mini-barcodes based on COI gene fragments provide an effective and accurate method for identification of pangolin species and authentication of pangolin scale products.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae).

BACKGROUND Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity.METHODS Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples.OBJECTIVES To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons.FINDINGS We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%).MAIN CONCLUSIONS Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

Molecular Identification of Pathogenic Bacteria in Kantong Semar Plants (Nepenthes Gracillis) Based on Mitochondrial 16S rRNA Gene

Nepenthes is a protected and unique carnivorous plant because it has a fluid-filled pouch at the end of the leaf that serves as an insect trap. The existence of Nepenthes gracillis in ecosystems is endangered, one of the reasons is caused by the attack of pathogenic bacteria, so it is necessary to identify pathogenic bacteria that attack N. gracillis as an effort to handle. The purpose of this study was to identify pathogenic bacteria in N. gracillis using 16S rRNA gene sequencing results. This study aims to identify the 16S rRNA sequence and phylogenetic of bacteria attached in N. Gracillis leaves, and the identification was confirmed using molecular data. The construction of phylogenetic topology was made based on ML and NJ method using Kimura-2 parameter model. Subsequently, molecular characterization and identification was conducted based on 720 bp of 16S rRNA gene similarity, sequence variation, genetic distance, and phylogenetic topology. According to the results, the bacteria species attached in N. Gracillis identified as Lelliottia nimipressuralis, and closely related with Erwinia aphidicola. Genetic distance between sample and related species was 0,0173, with 98,26% similarities, then strongly 100 bootstrap analyses. Thus, this study provides more information for Lelliottia nimipressuralis phylogenetic studies using different marker.

The success story of Labiobaetis Novikova & Kluge in the Philippines (Ephemeroptera, Baetidae), with description of 18 new species.

Material collected between 1994 and 2020 in the Philippines, covering most main islands like Luzon, Mindoro, Palawan, Negros, Cebu, Leyte, and Mindanao and some smaller islands, substantially increased our knowledge of Labiobaetis Novikova & Kluge in this archipelago. Only three species were previously reported: L. molawinensis (Müller-Liebenau, 1982) and L. sumigarensis (Müller-Liebenau, 1982) from larvae and L. boettgeri (Ulmer, 1924) from adults. Eighteen new species have been identified using a combination of morphology and genetic distance (COI, Kimura 2-parameter). They are described and illustrated based on their larvae and a key to all species in the Philippines is provided. The total number of Labiobaetis in the Philippines has increased to 21 species. Additional diversity of Labiobaetis based on molecular evidence only is presented as Molecular Operational Taxonomic Units (MOTUs) without description. The interspecific K2P distances in the Philippines are between 15% and 27%, the intraspecific distances are usually between 0% and 3%. The total number of Labiobaetis species worldwide is augmented to 144.

Genetic variation of E6 and E7 genes of human papillomavirus 52 from Central China.

Cervical cancer is the fourth most common malignant tumor in women worldwide and is closely related to human papillomavirus (HPV). Women have the highest susceptibility to HPV-52 type in Jingzhou, China. In this study, E6-E7 sequences of 183 HPV-52 positive samples were amplified by a polymerase chain reaction and sequenced. HPV-52 E6-E7 gene variations were analyzed. The phylogenetic tree was constructed using the Kimura 2-parameter method. The secondary structure of the protein was analyzed. The selective pressure to E6-E7 genes was estimated using PAML. In addition, the B cell epitopes of the E6-E7 sequences in HPV-52 were predicted by the ABCpred server. In E6 sequences, 15 single nucleotide variants were observed, including 6 nonsynonymous variants and 9 synonymous variants. In E7 sequences, 19 single nucleotide variants occurred, including 10 nonsynonymous variants and 9 synonymous variants. Sixamino acidvariants, including 3 nonconservative substitutions, were found in sequences encoding the alpha helix. Eight amino acid variants, including three nonconservative substitutions, occurred in sequences encoding the strand. Through phylogenetic analysis, the E6-E7 sequences were mainly distributed in B lineage. In HPV-52 E6-E7 sequences, no positively selected site was found. The nonconservative substitutions, such as K93R, K93E in E6, T37I, and D38N in E7, affected multiple hypothetical epitopes in the B cell. This study provides information for the investigation of HPV epidemic characters. The discovery of new variants of HPV-52 may lay the basis for the development of the virus diagnosis, further study of cervical cancer, and vaccine design in Central China.

HIV-1 diversity and compartmentalization in urine, semen, and blood.

HIV-1 persists indefinitely in multiple cellular reservoirs despite antiretroviral therapy. We previously demonstrated HIV-1 compartmentalization in kidney and urine. Here, we further characterized viruses in urine and when available, compared them to those present in semen from HIV-1 positive participants with detectable plasma viremia to further understand the viral dynamics in the upper and lower genitourinary tract.Blood and urine samples were obtained from 19 HIV-1 positive participants. Simultaneous semen samples were obtained from 16 of the 19 participants. HIV-1 envelope (env) gene sequences were obtained by single-genome amplification (SGA) and neighbor-joining trees were constructed using the Kimura 2-parameter model.HIV-1 env gene sequences were amplified from blood in 19/19 (100%) participants, urine in 18/19 (95%) participants, and semen in 12/16 (75%). In individuals from which both urine and semen samples were obtained, differences in viral shedding between the 2 sources were observed, where HIV-1 env sequences could only be amplified from either urine or semen. Longitudinal phylogenetic analysis of urine-derived env sequences from 1 participant demonstrated that urine clusters distinct from blood are maintained over time (20 weeks), consistent with viral compartmentalization and local replication. Comparison of urine and semen derived sequences demonstrated either virus compartmentalization or equilibration.Our results demonstrate that when present, viral compartmentalization in urine persists over time. Comparison of timing of viral shedding in urine and semen samples from our cohort suggest different viral kinetics between the upper and lower genitourinary tract and sequence analysis suggests that HIV-1 populations in urine and semen can either be imported from blood or produced locally.

Identification of Edible Fish Species of Pakistan Through DNA Barcoding

Fish is a fundamentally healthy food, loaded with essential nutrients, high protein content, vitamin D, and omega-three fatty acid. Mislabeling is a common problem in the fish industry that causes an imbalance in prices and fluctuation in the market. DNA barcoding is a potential technique for authentication of mislabeled and misidentified fish species. In this study, 11 freshwater and 6 marine fish species were used for DNA barcoding and further authentication using the mitochondrial cytochrome b (Cyto b) gene. Cyto b was amplified using PCR, producing an average read length of 1,141 bp. The obtained sequences were compared to the National Center for Biotechnology Information database using the Basic Local Alignment Search Tool. The average AT content (55.20%) was higher than the average GC content (44.78%) in marine and freshwater fish species. The mean genetic Kimura 2-parameter distances for species, genus, families, and orders were 0.311, 0.308, 0.023, and 0.337, respectively. Phylogenetic tree analysis revealed that most of the freshwater fish species clustered together due to the fact that they were in the same order or family, while the marine fish species clustered distantly. Single nucleotide polymorphism (SNP) analysis of all species in the study revealed distinct features regarding unique sites. All fish species could be identified based on their unique SNP profiles. Based on SNP data, DNA sequence based QR codes were developed for accurate identification of fish species. This is the first study to develop DNA-based QR barcodes for proper authentication of species during the chain of custody using simple technology.

A phylogenetic analysis of Crested Serpent Eagle (Spilornis cheela) based on cytochrome-c oxydase subunit I (COI): a stepping stone towards genetic conservation of raptors in Indonesia

As one of the most widespread raptors in Asia, Crested Serpent Eagle (Spilornis cheela) are genetically varied. However, due to the limited studies on genetic aspects, the genetic difference based its geographic distribution are difficult to define. A thorough phylogenetic analysis is needed to create a coherent taxonomic definition for the sake of conservation; starts with phylogenetic analysis based on cytochrome-c oxydase subunit I (COI) gene as the standardized genetic marker to define species. This research was aimed to resolve within species phylogenetic diversity and to explain the relationship between species based on COI gene. Total DNA was isolated from blood samples. The BLAST confirmed PCR amplified COI gene fragments were visualized on 0.8 agarose gel. Sequencing was carried out by FirstBase Laboratory Malaysia. Phylogenetic analysis was done using AMOVA and MEGA7 software in Neighbor Joining method with Kimura-2 parameter. Reconstructed phylogenetic tree confirmed the divergence between three samples and another S. cheela from other regions respective to their geographic distributions. There was 0,014 genetic distance between samples and S. cheela and 0,015 between S. cheela and other species among genus Spilornis. This research also revealed that S. cheela and S. holospilus were not significantly varied.

DNA barcode and phylogenetic analysis of dung beetles (Coleoptera: Scarabaeidae) from the Western Ghats biodiversity hotspot, India

Dung beetles are diverse, cosmopolitan, and endemic to different biogeographic zones. But, their taxonomy and phylogeny is currently not well understood in the old-world tropics. Within the subfamily Scarabaeinae, Onthophagus is the most species rich and complex genus with 2189 species worldwide. India has approximately 400 species of dung beetles belonging to 30 genera. Based on morphology, the dung beetles of certain genera in Oriental and Afrotropical regions have been classified into Species Groups. Presently the genetic information of dung beetles of India is poorly represented in the databases, thus limiting knowledge on the phylogenetic relationships of the Indian fauna. In this study, we aimed to provide barcodes of 27 species belonging to Onthophagus and one species of Onitis and to examine whether the resulting phylogenetic tree complies with the Species Groups. Kimura 2 parameter model was used to measure genetic distance and Neighbour Joining method was used for the tree construction. The tree topology did not fully support the Species Groups of Onthophagus as other studies from the African region have noted. The DNA barcodes generated from this study can serve as a reference for future phylogenetic studies.

Vietnamella chebalingensis, a new species of the family Vietnamellidae (Ephemeroptera) from China based on morphological and molecular data.

Vietnamella chebalingensis Tong, sp. nov. is described based on larval stage from China. The new species is distinguished from its congeners by the following combination of characters: body purple black with mottled white markings; length of inner pairs of cephalic projection approximately half of the outer pairs; posterior margin of abdominal tergites I-VI and VIII-X each with a pair of pointed projections, but tergite VII bearing only a single projection. The morphological evidence and molecular data (COI, Kimura 2-parameter) support that this new species represents the sixth species of the genus Vietnamella. An identification key to known larvae of the genus Vietnamella is provided.

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.