Barcoding Analysis Research Articles

Identification of Phalacronotus bleekeri (Günther, 1864) (Siluridae) From the Mekong Delta, Vietnam and Use of Morphological Analysis to Separate Populations

This study identified Phalacronotus to species level and examined its morphometric variation among three collection localities, and between sexes, in the Mekong Delta, Vietnam. Specimens were collected from local markets and fishers, including An Giang (n = 36) and Can Tho (n = 36) in the Hau River and Tien Giang (n = 30) in the Tien River for morphological measurements and DNA barcoding analysis. Eight mitochondrial cytochrome oxidase subunit I (COI) sequences of Phalacronotus sp. shared 99.6 ± 0.23 % similarity with five GenBank (GB) P. bleekeri sequences and differed by 7.6 % to 13.3 % from seven GB P. apogon sequences. The Vietnamese specimens of Phalacronotus were similar to P. bleekeri in number of anal fin rays (75–80) and the shape of the upper jaw teeth. Thus, the COI sequences and these morphological characteristics indicate that Phalacronotus sp. samples collected in the Mekong Delta are Phalacronotus bleekeri (Günther, 1864). Morphometric characteristics differed between populations in 15 of 19 indices (P < 0.05) and between females and males in three characteristics relating to the belly. Head characteristics, body depth, and caudal peduncle depth were the main parameters contributing to the inter-population variation. Based on size-adjusted data, 96.7 % of specimens were correctly classified from the Tien Giang population, while 27.8 % and 33.3 % were misclassified between the Can Tho and An Giang populations, respectively. These results indicate that morphometric parameters of P. bleekeri varied mainly between populations in the two Mekong River tributaries.

Phylogenetic analysis of black piper (Piper spp.) population collected in different locations of Viet Nam based on the ITSU1-4 gene region

The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses and it has been recommended as a core plant DNA barcode. To compare and find out the genetic diversity difference, some individuals Peper were collected in different localities in Vietnam when using the ITS of nuclear ribosomal DNA. The ITS gene region from the nuclear genomes was tested for suitability as DNA barcoding regions of thirty-nine Peper individuals. Universal primers were used and sequenced products were analyzed using the Maximum Likelihood method and Tamura-Nei model in the MEGA X program. We did not observe high variability in intraspecific distance within the ITSu1-4 gene region between individuals ranging from 0.000 to 0.155. The size of the gene region has fluctuated from 667 to 685 bp between different individuals with the percentage (G + C) contained in the ITSu1-4 gene region which ranged from 54.776% to 60.805%, mean = 60.174%. The values of Fu’s Fs, D, Fu and Li’s D* and F* were negative as well (Fs = -0.209, D = -1.824; p < 0.05, D* = -1.205; not significant, p > 0.10 and F* = -1.699; not significant, 0.10 > p > 0.05) indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred. The value of Strobeck’s S is high (S = 0.684). The results of evolutionary relationships of taxa obtained 3 groups with the highest value of Fst are shown in the pairs of groups II and III (Fst = 0.151) and the lowest is in groups II and I (Fst = 0.015). All of the new sequences have been deposited in GeneBank under the following accession numbers MZ636718 to MZ636756. This database is an important resource for researchers working on Species of Peper in Vietnam and also provides a tool to create ITSu1-4 databases for any given taxonomy.

Bar-HRM for Species Confirmation of Native Plants Used in Forest Restoration in Northern Thailand

Plant species confirmation is a crucial step in using native plant species for forest restoration. To enhance this, a hybrid method of DNA barcoding and high-resolution melting analysis (Bar-HRM) was investigated in this study. In total, 12 native plant species samples were collected from forest restoration sites in Nan, a province in Northern Thailand. Simulation HRM analysis was performed to find the most appropriate region for in vitro Bar-HRM analysis. After that, in vitro Bar-HRM was carried out to validate the performance of native plant species. Results from both simulation and in vitro analyses revealed that the nuclear ribosomal internal transcribed spacer (ITS) region can be used as a primer set that can clearly discriminate native plant species in this study. With our study, Bar-HRM was proved of use in native plant species confirmation, even if that species had no molecular data available. In this context, Bar-HRM would be useful for the identification of native plant species used in tropical forest restoration not only in Thailand but also in any areas with similar plant groups.

Genetic relationship and source species identification of 58 Qi-Nan germplasms of Aquilaria species in China that easily form agarwood.

Recently, Qi-Nan germplasm, the germplasm of Aquilaria species that easily forms agarwood, has been widely cultivated in Guangdong and Hainan Provinces in China. Since the morphological characteristics of Qi-Nan germplasm are similar to those of Aquilaria species and germplasm is bred by grafting, it is difficult to determine the source species of this germplasm by traditional taxonomic characteristics. In this study, we performed a DNA barcoding analysis of 58 major Qi-Nan germplasms as well as Aquilaria sinensis, A. yunnanensis, A. crassna, A. malaccensis and A. hirta with 5 primers (nuclear gene internal transcribed spacer 2 (ITS2) and the chloroplast genes matK, trnH-psbA, rbcL and trnL-trnF). This field survey in the Qi-Nan germplasm plantations in Guangdong and Hainan Provinces aimed to accurately identify the source species of Qi-Nan germplasm. According to the results, ITS2 and matK showed the most variability and the highest divergence at all genetic distances. This ITS2+matK combination, screened for with TaxonDNA analysis, showed the highest success rate in species identification of the Qi-Nan germplasm. Clustering in the phylogenetic trees constructed with Bayesian inference and maximum likelihood indicated that the Qi-Nan germplasm was most closely related to A. sinensis and more distantly related to A. yunnanensis, A. crassna, A. malaccensis and A. hirta. Therefore, this study determined that the source species of the Qi-Nan germplasm is A. sinensis.

Culicoides species community composition and feeding preferences in two aquatic ecosystems in northern Spain

BackgroundAquatic ecosystems provide breeding sites for blood-sucking insects such as Culicoides biting midges (Diptera: Ceratopogonidae), but factors affecting their distribution and host choice are poorly understood. A study was undertaken at two nature reserves in northern Spain to examine the abundance, species composition, population dynamics and feeding patterns of biting midges between 2018 and 2019.MethodsCulicoides were captured by light suction traps baited with CO2 and by sweep netting vegetation. Blood meals and species identification of blood-fed specimens were determined using cytochrome c oxidase I subunit (COI) DNA barcoding. Multivariate generalized linear models were used to evaluate the associations between the abundance of Culicoides, the species richness and other parameters.ResultsThe 4973 identified specimens comprised 28 species of Culicoides. These included two species reported for the first time in northern Spain, thus raising to 54 the number of Culicoides species described in the region. Specimens of all 28 species and 99.6% of the total specimens collected were caught in suction traps, while sweep netting vegetation revealed just 11 species and 0.4% of the total specimens. Midge abundance peaked in June/early July, with five species comprising > 80% of the captures: Culicoides alazanicus (24.9%), Culicoides griseidorsum (20.3%), Culicoides poperinghensis (16.2%), Culicoides kibunensis (10.7%) and Culicoides clastrieri (9.6%). DNA barcode analysis of blood meals from eight Culicoides species revealed that they fed on 17 vertebrate species (3 mammals and 14 birds). Species in the subgenus Avaritia were primarily ornithophilic, except for C. griseidorsum and C. poperinghensis. Host DNA from blood meals was successfully amplified from 75% of blood-fed females. A pictorial blood meal digestion scale is provided to accurately assess the blood-fed status of female Culicoides.ConclusionsThe large number of different blood meal sources identified in the midges captured in this study signals the likely importance of wild birds and mammals (e.g. red deer and wild boar) as reservoir/amplifying hosts for pathogens. Available hosts are more exposed to being bitten by biting midge populations in aquatic ecosystems in late spring and early summer.

First record of <i>Eptesicus furinalis</i> (Chiroptera, Vespertilionidae) from the state of Maranhão, Brazil

A specimen of Eptesicus furinalis was collected in the municipality of Cândido Mendes, in the state of Maranhão, Brazil. It was a non-lactating adult female, with dark chestnut dorsal coloration, yellow venter, hairless membranes, short and rounded ears, and a pointed tragus. The analysis of the DNA barcode of the COI mitochondrial gene revealed a 99.80 % similarity with the sequence of E. furinalis deposited in the BOLDSystems platform. The combined analysis of the morphological and molecular data confirmed the occurrence of E. furinalis in the state of Maranhão. This extends the known distribution of the species 676.1 km from the nearest recorded locality in the Ceará State.

Genetic diversity of wild zebrafish Danio rerio populations available in Bangladesh

The morphometric and genetic variations of 4 wild populations of zebrafish ( Danio rerio ) in Bangladesh were studied using DNA barcoding and RAPD (Random Amplified Polymorphic DNA). The zebrafish populations were as ZMS (zebrafish from the Brahmaputra river, Mymensingh), ZFP (zebrafish from the Kumar river, Faridpur), ZKH (zebrafish from the Moyurriver, Khulna) and ZMG (zebrafish from the Aarial Beel, Munshiganj). The morphometric variations were insignificant to establish the genetic structure and diversity of these populations. Therefore, the genetic variation was analyzed with DNA barcoding and RAPD techniques. FishF1 and FishR1 primers were used for barcoding with amplification of Cytochrome Oxidase C subunit 1(COI) gene region and OPA03, OPA04 and OPF01 primers were used for RAPD analysis. In DNA barcoding analysis, the database provided species matches of 100% sequence similarity for these 4 wild fish populations with D. rerio . The overall sequence alignment similarity among these populations was 99.40%. The neighbor-joining phylogenetic tree was constructed from these COI barcode sequences also revealed genetic relationships and diversity among these populations. In RAPD analysis, a total number of 112 bands were found where 22 bands were polymorphic, indicated 19.64% polymorphism among these 4 wild populations. The percent polymorphic loci and genetic distance varied within a range of 14.00–26.67 and 0.2877–1.6094 respectively. Nei's genetic similarity among these 4 populations across all the primers ranged from 0.2000 to 0.7500. The Dendrogram based on Nei's genetic distance showed 3 clusters indicating the ZFP population is the maximum genetically diversified among them however; the ZMS and ZKH populations were in the same cluster. The knowledge of the molecular variation of wild zebrafish could be helpful for planning more effective and efficient managing strategies for this model species and also provide a significant contribution to zebrafish research all over the world.

Clarifying the identity of two resembling hoverfly species, Betasyrphus serarius and B. nipponensis (Diptera: Syrphidae: Syrphini), based on morphology and DNA barcoding

• Identity of resembling Betasyrphus serarius and B. nipponensis is investigated. • Additional diagnostic characters are proposed for B. serarius and B. nipponensis. • Betasyrphus nipponensis is recorded for the first time in Korea. • Nine available Betasyrphus species are recognized as a robust barcode cluster. • Australian, Pakistan and Malaysian barcodes as distinct species near serarius. The two Palaearctic members of the hoverfly genus Betasyrphus , B. serarius (Wiedemann, 1830) and B. nipponensis (Goot, 1964), closely resemble each other, except for their male genitalic characters. For this reason, these two species have been often mistaken for each other. In Korea, only B. serarius has been recognized as a sole species based on mixed specimens from these two species. In the present study, we attempted to define B. serarius and B. nipponensis more precisely and identify additional diagnostic characteristics to distinguish these species. We examined external morphological characters including the genitalic structures, the wing microtrichia distributions (WMDs), as well as the wing interference patterns (WIPs). In addition, we analyzed the DNA barcode sequences of individuals, covering as many morphological variants as possible, to distinguish the females of these two species. As a result, we found some additional diagnostic male genitalic and WMD characters. Unfortunately, despite our efforts, females of these two species remain undistinguishable. DNA barcoding analysis did not reveal any significant differences between B. serarius and B. nipponensis . Our analysis based on 20 Korean samples and 486 Syrphidae sequences retrieved from BOLD Systems (ver. 4) and GenBank showed that the nine available Betasyrphus species including the type species formed a robust barcode cluster. Betasyrphus serarius and B. nipponensis together with three unidentified species from Pakistan, Australia and Malaysia also formed a strong sub-cluster. In addition, we provide an identification key, diagnoses, and descriptions with color photographs.

The <i>COI</i> haplotype diversity of the pelagic polychaete <i>Tomopteris</i> (Annelida: Tomopteridae) collected from the Pacific coast off Kii Peninsula, central Japan

The polychaete genus Tomopteris is an important component of the zooplankton and is also known for its communication using bioluminescence. However, there have been few studies about the species diversity and ecology of Tomopteris because of taxonomic and handling difficulties resulting from the fragility of their bodies. In this study, we performed DNA barcoding and species delimitation analysis to better understand the species diversity of Tomopteris using 17 specimens collected on a research cruise conducted off the eastern Kii Peninsula, Japan. Due to damage to the detailed morphological characteristics necessary for identifications based on the current taxonomy, none of the specimens was morphologically identifiable at the species level. The analyses of the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear large ribosomal subunit (28S) RNA gene sequences revealed 7–8 molecularly delimited species among them, which was greater than the number of species currently recognized in Japan. These results suggest that the traditional morphology-based taxonomy overlooks the taxonomic complexity of Tomopteris, thus a combinatory analysis of both morphology and DNA barcoding will be desirable for assessing the true species biodiversity of Tomopteris.

Biodiversity of Duckweed (Lemnaceae) in Water Reservoirs of Ukraine and China Assessed by Chloroplast DNA Barcoding

Monitoring and characterizing species biodiversity is essential for germplasm preservation, academic studies, and various practical applications. Duckweeds represent a group of tiny aquatic plants that include 36 species divided into 5 genera within the Lemnaceae family. They are an important part of aquatic ecosystems worldwide, often covering large portions of the water reservoirs they inhabit, and have many potential applications, including in bioremediation, biofuels, and biomanufacturing. Here, we evaluated the biodiversity of duckweeds in Ukraine and Eastern China by characterizing specimens using the two-barcode protocol with the chloroplast atpH–atpF and psbK–psbI spacer sequences. In total, 69 Chinese and Ukrainian duckweed specimens were sequenced. The sequences were compared against sequences in the NCBI database using BLAST. We identified six species from China (Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna minor, Lemna turionifera, and Wolffia globosa) and six from Ukraine (S. polyrhiza, Lemna gibba, Lemna minor, Lemna trisulca, Lemna turionifera, and Wolffia arrhiza). The most common duckweed species in the samples from Ukraine were Le. minor and S. polyrhiza, accounting for 17 and 15 out of 40 specimens, respectively. The most common duckweed species in the samples from China was S. polyrhiza, accounting for 15 out of 29 specimens. La. punctata and Le. aequinoctialis were also common in China, accounting for five and four specimens, respectively. According to both atpH–atpF and psbK–psbI barcode analyses, the species identified as Le. aequinoctialis does not form a uniform taxon similar to other duckweed species, and therefore the phylogenetic status of this species requires further clarification. By monitoring duckweeds using chloroplast DNA sequencing, we not only precisely identified local species and ecotypes, but also provided background for further exploration of native varieties with diverse genetic backgrounds. These data could be useful for future conservation, breeding, and biotechnological applications.

The State of Beach Litter Pollution during the COVID-19 Pandemic: A Case Study of the Israeli Coasts

This research examines the impact of the COVID-19 pandemic on marine litter pollution along the Israeli coast, which constitutes a terminal area in the Mediterranean Sea large-scale circulation. Surveys for litter were conducted on four beaches along the Israeli Mediterranean coast. Sampling was done pre-pandemic in June, September, and December of 2019; during the first year of the pandemic in March, June, September, and December of 2020; and during the second year of the pandemic in March 2021. The study revealed that during the first year of the pandemic, beach litter concentration was reduced by 47 ± 27% on all beaches. The decrease in beach litter continued during the second year on most beaches (34 ± 22% reduction) and was most observable for plastic products, which have a high potential to float and travel long distances. The decline in beach litter concentration in the Israeli beaches during the pandemic was statistically significant. The barcode analysis indicates the appearance of non-Israeli debris on the shores (22-81% pre-COVID-19 and 5-47% during-COVID-19), which supports the assumption that the reduction in marine litter pollution was affected by the decline in beach activity both locally and in other coastal neighboring countries. An additional effect of the pandemic was the appearance of single-use plastic masks and gloves in April 2020, influenced by the epidemic’s regulations and restrictions. Despite the decline in beach marine litter concentration during COVID-19, plastic pollution is a high concern in this heavily populated coastal region. Our data set provides an indication for the potential of local and regional regulations to reduce further beach litter pollution and a baseline for continuous monitoring of these processes. Highlights The impact of COVID-19 on beach litter pollution along Israel’s coast was studied Significant decline in beach litter concentration during the Covid-19 pandemic. Barcode analysis reveals debris is 22-57% from non-local sources. Litter associated with COVID-19 was identified for the first time just as pandemic start

Character-based Diagnostic Keys, Molecular Identification and Phylogenetic Relationships of Fishes based on Mitochondrial Gene from Pulicat Lake, India: A Tool for Conservation and Fishery Management Purposes

Background: Pulicat Lake is one of India’s most valuable ecosystems. Correct species identification is essential for the conservation and management of finite resources. Molecular signatures and character-based keys could be used for faster identification. This will be a powerful tool to obtain a significant amount of accurate information quickly and conveniently and could be used for forensic applications and the conservation of fish in Indian waters. Methods: In addition to morphological identification, the species were identified using various approaches, such as Neighbour-joining (NJ), Maximum Likelihood (ML) trees and the Character-based method. For each species, the LOGic (BLOG 2.0) DNA Barcode analysis method was used to identify different positions of key diagnostic nucleotides in a given set. Result: DNA barcodes were generated from twenty-nine species of fish representing seven orders, 26 genera and 21 families in this study. The current study’s findings show that the 16S rRNA barcodes facilitated successful fish species identification and also provided phylogenetic information to differentiate the fishes. The character-based molecular diagnostic keys for fishes were also developed in-silico from the data set comprising 393 mitochondrial 16S rRNA sequences including the sequences generated in the present study and other published resources.

Plant genetic diversity by DNA barcoding to investigate propolis origin

Identify the botanical origins of a certain type of propolis may be challenging and time demanding, since it involves bee's behavior observation, plant resins collection and chemical analysis. Thus, this study aimed to determine the plant genetic materials in propolis from southern Brazil using the DNA barcoding to investigate their botanical origins, as well as to compare it with the phytochemical composition determined by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and with the pollinic profile. As principal results, non-native Populus carolinensis Moench (Salicaceae) was almost the only DNA source in some propolis samples, which coincided with the presence of flavonoids typical from poplar exudates. Conversely, other propolis samples had DNA material coming mainly from native plant species, most of them characterized to the species level, although no specific chemical markers from those plants could be identified by UHPLC-HRMS. However, pollen from several plants identified by the DNA barcoding were extracted from some propolis samples. Despite the identification of typical diterpenes, DNA material from Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae), which have been indicated as a major resin source for propolis from preservation areas in southern Brazil, was found in very small abundancies, likely because bees do not drag tissue material containing DNA when collecting resin from this native species. In conclusion, DNA barcoding analysis successfully provided information about the provenance of propolis, although, depending on the plant resin sources, this information is likely to come from pollen.

DNA barcoding of Philippine freshwater eels of families Anguillidae and Synbranchidae

The study used morphological and genetic approach for the DNA barcoding analysis of the inter- and intra-species relationships among catadromous Philippine freshwater eels. Past DNA barcoding studies on Philippine anguillid eels came from Northern Luzon. This study aimed to determine the DNA barcodes of the freshwater eels of the Philippines using the cytochrome c oxidase 1 (CO1) gene of the mitochondrial DNA. Specimens were collected from six sites in the Philippines. Four Anguilla species – Anguilla bicolor pacifica Schmidt, 1928, A. celebesensis Kaup, 1856, A. interioris Whitney, 1938 and A. marmorata Quoy & Gaimard, 1824 and one Monopterus species– Monopterus javanensis Lacepéde, 1800 were collected and identified. Morphological features varied from the taxonomic guides for Anguilla celebesensis and A. interioris with their body color and fin length. Genetic divergence estimates using Kimura 2-parameter substitution model showed an intraspecific variation of 0–0.4% and interspecific variation of 4.1– 27.6%. The ML tree generated was similar to the previous studies and indicated the monophyly of the Indo–Pacific freshwater anguillid eel lineage. This study also reports the first genetic record of M. javanensis sampled from Batac, Ilocos Norte, Philippines.

Morphological description and DNA barcoding study of Paramignya trimera collected in Khanh Hoa, Vietnam

Paramignya trimera is a valuable traditional medicinal plant, typically distributing in Khanh Hoa, Vietnam. In recent years, P. trimera has been exploited to treat liver diseases such as hepatitis, cirrhosis and liver cancers. The recorded during the investigation, in Khanh Hoa currently appears a number of species with similar phenotypes known collectively as P. trimera and appeared some morphology with differences in leaf morphology and thorns. Therefore, this study was carried out for identification, breeding and propagation conservation.This study has been described, characterized morphological characteristics and analyzed DNA barcodes based on ITS, matK and rbcL gene markers. About the morphology, P. trimera is distinguished from other species of the genus Paramignya with the following characteristics: elongated, single leaf, leaf-shaped lobe end, leaf veins shaped like a feather. Petiole with big, pointed and straight spines. Corolla 3, 6 stamens, upper election, with three sepals and two ovule leaves, the seed has a seed coat. The analysis of DNA barcodes shows that the results combination of the analysis of the three regions of ITS, matK and rbcL can be used to determine the genetic relationship between five samples of P. trimera. The nucleotide sequence lengths of the samples for the three regions of the ITS, matK and rbcL genes are 850 bp, 850 bp and 500 bp, respectively. On the basis of phenotypic tree analysis, the samples (X1, X2, X3, X4, X5) have the same evolutionary origin in ITS, matK and rbcL. Base on to identified the five morphological forms of P. trimera distributed in Khanh Hoa as the same species and expanded the distribution area as well as in-depth research on P. trimera morphologies in Khanh Hoa, Vietnam.

First record of blacknape large-eye bream Gymnocranius satoi (Perciformes: Lethrinidae) in the Philippines

The Philippines has been regarded as the center of the center of marine shorefish biodiversity, having the highest number of fish species per square area in the world. The blacknape large-eye bream, Gymnocranius satoi, has been reported to occur from Southern Japan, Taiwan to Northwestern Australia and to the Coral Sea, but has not previously been recorded from the Philippines. From 2011 – 2019, the National Fisheries Research and Development Institute (NFRDI) collaborated with the National Museum of Natural History of the Smithsonian Institution (NMNH/SI), USA, and the Old Dominion University (ODU), Virgina, USA, to inventory all commercial fish species sold in fish markets around the Philippines. During three (3) fish market surveys (Dumaguete City Market, Negros Oriental; Claveria Public Market, Cagayan, Northern Luzon; and Tabaco City Market, Albay, Southeastern Luzon in 2013, 2016 and 2017, respectively), we collected and eventually identified using morphological and DNA barcoding (COI) analysis, seven (7) specimens of G. satoi, representing the first records of this species from the country. Since the potential to discover new species and first records of fish species in the Philippines is high, further taxonomic study of the genus Gymnocranius is needed.

Are we there yet? Benchmarking low-coverage nanopore long-read sequencing for the assembling of mitochondrial genomes using the vulnerable silky shark Carcharhinus falciformis

BackgroundWhole mitochondrial genomes are quickly becoming markers of choice for the exploration of within-species genealogical and among-species phylogenetic relationships. Most often, ‘primer walking’ or ‘long PCR’ strategies plus Sanger sequencing or low-pass whole genome sequencing using Illumina short reads are used for the assembling of mitochondrial chromosomes. In this study, we first confirmed that mitochondrial genomes can be sequenced from long reads using nanopore sequencing data exclusively. Next, we examined the accuracy of the long-reads assembled mitochondrial chromosomes when comparing them to a ‘gold’ standard reference mitochondrial chromosome assembled using Illumina short-reads sequencing.ResultsUsing a specialized bioinformatics tool, we first produced a short-reads mitochondrial genome assembly for the silky shark C. falciformis with an average base coverage of 9.8x. The complete mitochondrial genome of C. falciformis was 16,705 bp in length and 934 bp shorter than a previously assembled genome (17,639 bp in length) that used bioinformatics tools not specialized for the assembly of mitochondrial chromosomes. Next, low-pass whole genome sequencing using a MinION ONT pocket-sized platform plus customized de-novo and reference-based workflows assembled and circularized a highly accurate mitochondrial genome in the silky shark Carcharhinus falciformis. Indels at the flanks of homopolymer regions explained most of the dissimilarities observed between the ‘gold’ standard reference mitochondrial genome (assembled using Illumina short reads) and each of the long-reads mitochondrial genome assemblies. Although not completely accurate, mitophylogenomics and barcoding analyses (using entire mitogenomes and the D-Loop/Control Region, respectively) suggest that long-reads assembled mitochondrial genomes are reliable for identifying a sequenced individual, such as C. falciformis, and separating the same individual from others belonging to closely related congeneric species.ConclusionsThis study confirms that mitochondrial genomes can be sequenced from long-reads nanopore sequencing data exclusively. With further development, nanopore technology can be used to quickly test in situ mislabeling in the shark fin fishing industry and thus, improve surveillance protocols, law enforcement, and the regulation of this fishery. This study will also assist with the transferring of high-throughput sequencing technology to middle- and low-income countries so that international scientists can explore population genomics in sharks using inclusive research strategies. Lastly, we recommend assembling mitochondrial genomes using specialized assemblers instead of other assemblers developed for bacterial and/or nuclear genomes.

An unexpected species complex unveiled in southern European populations of Phragmatiphila nexa (Hbner, [1808]) (Lepidoptera, Noctuidae, Noctuinae, Apameini).

DNA barcoding analyses of Phragmatiphila nexa (Hbner, 1808) populations unveiled an unexpected divergence in mtDNA of Italian populations, showing the existence of three allopatric cryptic species. The northernmost BIN is shared with specimens from most other European countries, the southernmost one includes specimens from Basilicata and Calabria regions, and the last BIN includes specimens from Apennines, Sardinia and Corsica. Wing pattern as well as male and female genitalia support the existence of three different species along the Italian peninsula: Phragmatiphila nexa north of the Po River for which we designate a neotype, Phragmatiphila insularis (Turati, 1913), stat. rev. in the Apennines as well as in Sardinia (and Corsica), and Phragmatiphila parenzani sp. n. in the south. The Italian distribution of the genus Phragmatiphila is presented in detail.

Identification of the original plants of cultivated Bupleuri Radix based on DNA barcoding and chloroplast genome analysis.

Bupleuri Radix is the dry root of certain species of the genus Bupleurum and is commonly used in traditional Chinese medicine. The increasing global demand for Bupleuri Radix cannot be fulfilled with wild populations only. Therefore, cultivated Bupleurum is now the main commercial source of this medicinal product. Different species of Bupleurum show different medicinal properties and clinical effects, making reliable authentication and assignment of correct botanical origin for medicinal species critical. However, accurate identification of the cultivated Bupleurum species is difficult due to dramatic morphological variations resulting from cultivation. In this study, we sampled 56 cultivated Bupleurum populations of six different morphotypes (Types A-F) from the main production areas of China, and 10 wild populations of four species were used as reference materials. Conventional DNA barcoding was conducted to identify cultivated Bupleurum species. Additionally, verification based on complete chloroplast genomes was performed and new chloroplast markers were developed and evaluated. The combination of these methods resulted in the successful identification of all cultivated Bupleurum individuals. Three chloroplast regions are recommended as additional barcodes for the genus: ycf4_cemA, psaJ_rpl33, and ndhE_ndhG. This is a reliable and promising strategy that can be applied to the authentication of natural products and the identification of other medicinal plant species with similar taxonomic problems.

Filter-Feeding Bivalve Weakens Food Competition Between Crustaceans (Portunus trituberculatus, Marsupenaeus japonicus) in Integrated Culture Ponds: Evidence From 18S rDNA Barcoding and Stable Isotope Analysis

In order to investigate the effects of razor clams (Sinonovacula constricta) on the food composition and isotopic niches of swimming crabs (Portunus trituberculatus) and kuruma shrimp (Marsupenaeus japonicus) in polyculture systems, this study analyzed 60 P. trituberculatus, 60 M. japonicus and 30 S. constricta to quantify the food sources, food source contributions, and isotopic niches of cultured organisms using 18S rDNA barcoding and stable isotope techniques. The results were as follows: (1) In the P. trituberculatus-M. japonicus (PM) polyculture system, the Sobs and Shannon-Wiener indices of the stomach contents of P. trituberculatus and M. japonicus were not significantly different (P > 0.05). In the P. trituberculatus-M. japonicus-S. constricta polyculture (PMS) system, the Sobs and Shannon-Wiener indices of the stomach contents of P. trituberculatus and M. japonicus were also not significantly different (P > 0.05), but the Sobs indices of P. trituberculatus in the PMS system were significantly higher than those in the PM system (P < 0.05), M. japonicus shows a similar pattern. (2) 18S rDNA barcoding analysis showed the dominant taxa in the stomach contents of both P. trituberculatus and M. japonicus in the PM system were Trebouxiophyceae, Embryophyta and Rotifera, and the food overlap between them was 0.8992, which was significant (Q > 0.6). In the PMS system, the dominant taxa in the stomach contents of P. trituberculatus were Chrysophyceae, Intramacronucleata, and Embryophyta, and in M. japonicus were Chrysophyceae, Embryophyta, and Bacillariophyceae, in this system the food overlap was 0.2061, which was not significant (Q < 0.6). (3) Stable isotope analysis suggested, in both systems, the main food sources of P. trituberculatus and M. japonicus were iced trash fish, zooplankton, phytoplankton, and organic particulate matter (POM). Iced trash fish accounted 77.67% of food sources for P. trituberculatus and 69.42% for M. japonicus in the PM system, and 60.82% and 57.60% in the PMS system. (4) The isotopic niche overlap between P. trituberculatus and M. japonicus was 5.69% in the PM system and 1.21% in the PMS system. These results suggested food competition between P. trituberculatus and M. japonicus, and S. constricta can reduce the competition and isotopic niche overlap, improve the contribution of food sources such as phytoplankton. Razor clams also serve to purify the water and improve the utilization of iced trash fish by filtering phytoplankton (51.10%), POM (32.25%), SOM (7.47%), and iced trash fish (9.18%). Thus, P. trituberculatus-M. japonicus-S. constricta is a healthy and sustainable culture model.