Cytochrome C Oxidase I Sequences Research Articles

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective: To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles (An.) subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2 (ITS2) and cytochrome C oxidase I (COI) sequences deposited at the GenBank. Methods: Seventy-five ITS2, 210 COI and 26 concatenated sequences available in the NCBI database were used. Phylogenetic analysis was performed using Bayesian likelihood trees, whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis. Genetic diversity indices and genetic differentiation were also calculated. Results: Two genetically divergent molecular forms of An. subpictus species complex corresponding to sibling species A and B are established. Species A evolved around 37-82 million years ago in Sri Lanka, India, and the Netherlands, and species B evolved around 22-79 million years ago in Sri Lanka, India, and Myanmar. Vietnam, Thailand, and Cambodia have two molecular forms: one is phylogenetically similar to species B. Other forms differ from species A and B and evolved recently in the above mentioned countries, Indonesia and the Philippines. Genetic subdivision among Sri Lanka, India, and the Netherlands is almost absent. A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances. Genetic diversity indices reveal the presence of a long-established stable mosquito population, at mutation-drift equilibrium, regardless of population fluctuations. Conclusions: An. subpictus species complex consists of more than two genetically divergent molecular forms. Species A is highly divergent from the rest. Sri Lanka and India contain only species A and B.

First Report of Globisporangium (Pythium) mastophorum causing Damping-off / Root Rot on Parsley in Slovenia.

Pythium-like species cause damping-off symptoms of various hosts, including umbelliferous crops. In April 2023, parsley plantlets (Petroselinum crispum), showing stunted growth, yellowing, decayed roots and damping-off, were obtained from a nursery in central Slovenia, where parsley was grown in polystyrene trays in a greenhouse. Nearly 30% of plants were symptomatic. Sampled roots of ten plants contained ornamented oogonia (avg. 33.3 ± 1.4 µm in diam) with conical projections (5.2 ± 0.5 µm long) (Figure S1 A, B) in microscopically analyzed squash mounts. The pathogen was isolated from root pieces treated for surface disinfection with 0.5% sodium hypochlorite for 30 s, and washed with sterile water. Four 1-2 mm root pieces were taken from each of 10 plants, plated on the selective medium P5ARP, and incubated at 21 °C. Mycelia emerging from root pieces were transferred to carrot piece agar (CPA). Twenty-two equally looking oomycetous colonies were obtained; all sampled plants were infested. Oogonia formed by all colonies were similar to those observed on decayed roots and suggested that Globisporangium (Pythium) mastophorum is the causal disease agent. Analyses of partial β-tubulin (Kroon et al. 2004) and mitochondrial cytochrome c oxidase I (COI) gene sequences (Robideau et al. 2011) confirmed the identification. Obtained COI (Genbank accession number OR725417) sequence was 100% identical to that from G. mastophorum strain CBS 375.72 (EU350523), whereas the β-tubulin sequence (OR725416) corresponded to 99.6 % pairwise identity (KJ595502). Further, pathogenicity of an obtained isolate was tested on 4 wk-old curly leaf (cv. Petra F1) parsley. Half of a 7 d-old CPA culture, consisting of mycelium and oogonia, was finely cut and mixed with ca 50 ml of nonsterile commercial substrate (Potgrond H, AGRO-FertiCrop) in each of six 400 ml pots. Pots were filled with ca 300 ml additional substrate, into which 5 parsley seedlings were planted. Control plants were treated equally but with sterile CPA. Plantlets were watered with sterile tap water and held at ambient light conditions and temperature (night 18 °C - day 23 °C). After 14 d, inoculated plants started wilting and yellowing and showed stunted growth. After 21 d, roots were severely decayed and the seedlings damped-off (Figure S1 C). Four pieces each from 10 decayed roots were plated. Thirty-one pieces revealed pythium-like colonies. Obtained isolates were morphologically identical to the strain used for inoculation and identified as G. mastophorum. Control plants developed no foliar or root symptoms and no pythium-like species was obtained. Agricultural advisors observed occurrence of parsley damping-off also in other nurseries in Slovenia what may lead to spreading the pathogen to parsley in production fields and private gardens. The case emphasizes the need for implementing phytosanitary measures in order to eliminate primary inoculum. Reports from field-infected plants showed that G. mastophorum is a pathogen of parsley in Australia (Petkowski et al. 2013) and the USA (Tsuchida et al. 2018), and celery in the Czech Republic (Šafránková and Holková 2017). Others isolated G. mastophorum from parsley in The Netherlands (online database of the Westerdijk Fungal Biodiversity Institute, strain CBS 243.86). However, the here described case is, to the best of our knowledge, one of the rare documentations of damping-off due to G. mastophorum in Europe (Šafránková and Holková 2017) and the first in Slovenia. Funding: The work was funded by the Ministry of Agriculture, Forestry and Food of Slovenia, and Slovenian Research and Innovation Agency (ARIS Programs P4-0431 and P4-0072).

Molecular detection of blood protozoa and identification of black flies of the Simulium varicorne species group (Diptera: Simuliidae) in Thailand

Species of the Simulium varicorne group in Thailand have veterinary significance as vectors of haemosporidian parasites. Accurate identification is, therefore, critical to the study of vectors and parasites. We used morphology and molecular markers to investigate cryptic genetic lineages in samples identified as Simulium chumpornense Takaoka & Kuvangkadilok, 2000. We also tested the efficiency of the nuclear internal transcribed spacer 2 (ITS2) marker for the identification of species in this group. Morphological examinations revealed that S. chumpornense lineage A is most similar to S. khelangense Takaoka, Srisuka & Saeung, 2022, with minor morphological differences. They are also genetically similar based on mitochondrial cytochrome c oxidase I (COI) sequences. Geographically, the sampling site where paratypes of S. khelangense were originally collected is <50 km from where S. chumpornense lineage A was collected. We concluded that cryptic lineage A of S. chumpornense is actually S. khelangense. COI sequences could not differentiate S. kuvangkadilokaePramual & Tangkawanit, 2008 from S. chumpornense and S. khelangense. In contrast, ITS2 sequences provided perfect accuracy in the identification of these species. Molecular analyses of the blood protozoa Leucocytozoon and Trypanosoma demonstrated that S. khelangense carries L. shoutedeni, Leucocytozoon sp., and Trypanosoma avium. The Leucocytozoon sp. in S. khelangense differs genetically from that in S. asakoae Takaoka & Davies, 1995, signaling the possibility of vector-parasite specificity.

DNA Barcoding Analysis of Trinidad Haemagogus Mosquitoes Reveals Evidence for Putative New Species.

Background: Haemagogus janthinomys is a primary sylvan vector of yellow fever virus and the emerging Mayaro virus. However, despite its medical importance, there is a dearth of data on the molecular taxonomy of this mosquito species. Methods: In this study, DNA barcoding analysis was performed on 64 adult female mosquitoes from Trinidad morphologically identified as Hg. janthinomys. The mitochondrial cytochrome c oxidase I (COI) gene and ribosomal DNA internal transcribed spacer 2 (ITS2) region of the mosquitoes were PCR amplified and sequenced, and molecular phylogenies inferred. Results: The BLASTN analysis showed that only 20% (n = 13/66) of COI sequences had high similarity (>99% identity) to Hg. janthinomys and the remaining sequences had low similarity (<90% identity) to reference GenBank sequences. Phylogenetic analysis of COI sequences revealed the presence of four strongly supported groups, with one distinct clade that did not align with any reference sequences. Corresponding ITS2 sequences for samples in this distinct COI group clustered into three clades. Conclusions: These molecular findings suggest the existence of a putative new Haemagogus mosquito species and underscore the need for further, more in-depth investigations into the taxonomy and classification of the Haemagogus genus.

Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

COI metabarcoding better reveals the seasonal variations in the zooplankton community in the western Pacific Warm Pool

The environment of the western Pacific Warm Pool is considered to be pronouncedly uniform and stable compared with other tropical ocean environments. Knowledge of the seasonal variations in the zooplankton community structure in this region with a high plankton species diversity remains limited. In this study, three methods, including identification using microscopy, ZooScan digital imaging, and DNA metabarcoding, were used to analyze zooplankton samples collected in three seasons, and to detect the seasonal variations in the community. The results show that the metabarcoding method based on the mitochondrial cytochrome c oxidase I (COI) marker detected up to 2.3 times more zooplankton taxa than identification using microscopy. In contrast, the small subunit ribosomal RNA (18S) marker revealed fewer taxa than microscopy. Significantly positive correlations were observed between the sequence counts and three types of quantitative data, including the biovolume, dry weight biomass, and carbon biomass in all three seasons. The COI sequence counts exhibited a stronger correlation with the dry weight biomass than the biovolume and carbon biomass. Although both the microscopic and molecular methods detected seasonal variability of the zooplankton community, the COI metabarcoding method was more sensitive in detecting seasonal variations in the community, which was positively correlated with the taxonomic resolution of the different methods. In summary, the results of this study suggest that COI metabarcoding is an ideal tool for monitoring the seasonal variations in the diversity and community structure of zooplankton in the tropical open ocean regions with a stable environment all year round.

Overcoming taxonomic challenges in DNA barcoding for improvement of identification and preservation of clariid catfish species.

DNA barcoding without assessing reliability and validity causes taxonomic errors of species identification, which is responsible for disruptions of their conservation and aquaculture industry. Although DNA barcoding facilitates molecular identification and phylogenetic analysis of species, its availability in clariid catfish lineage remains uncertain. In this study, DNA barcoding was developed and validated for clariid catfish. 2,970 barcode sequences from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes and D-loop sequences were analyzed for 37 clariid catfish species. The highest intraspecific nearest neighbor distances were 85.47%, 98.03%, and 89.10% for COI, Cytb, and D-loop sequences, respectively. This suggests that the Cytb gene is the most appropriate for identifying clariid catfish and can serve as a standard region for DNA barcoding. A positive barcoding gap between interspecific and intraspecific sequence divergence was observed in the Cytb dataset but not in the COI and D-loop datasets. Intraspecific variation was typically less than 4.4%, whereas interspecific variation was generally more than 66.9%. However, a species complex was detected in walking catfish and significant intraspecific sequence divergence was observed in North African catfish. These findings suggest the need to focus on developing a DNA barcoding system for classifying clariid catfish properly and to validate its efficacy for a wider range of clariid catfish. With an enriched database of multiple sequences from a target species and its genus, species identification can be more accurate and biodiversity assessment of the species can be facilitated.

DNA barcodes of Saudi Arabian birds: Implications for species identification and diversity analysis

ObjectivesDNA barcoding using cytochrome c oxidase I (COI) gene is an effective tool for species identification with additional power of measuring molecular diversity and phylogenetic inference. In this study, we evaluated the performance of COI gene sequences for species identification and molecular diversity analysis of some Saudi Arabian birds. MethodsWe sequenced the 694 base pair segment of COI gene from 5 samples of white cheeked bulbul (Pycnonotus leucogenys), 4 samples of black scrub robin (Cercotrichas podobe), and 2 samples of crested lark (Galerida cristata), all belonged to the same Order. We also included all the COI sequences available in the GenBank for these birds with the aim of studying molecular diversity across geographies. For species identification, we enriched our dataset by including COI barcodes of other Saudi Arabian bird species, including Arabian partridge (Alectoris melanocephala), houbara bustard (Chlamydotis undulata macqueenii), green bee-eater (Merops orientalis), laughing dove (Streptopelia senegalensis), namaqua dove (Oena capensis) and collared dove (Streptopelia decaocto), representing different genera. ResultsWhite-cheeked bulbul from Saudi Arabia showed more diversity than the same birds from Iraq. The mean nucleotide difference and nucleotide diversity were 5.20 and 0.009 respectively with as Tajima’s D score of 1.6549 indicating the scarcity of rare alleles. The specimens of black scrub robin from Saudi Arabia and Djibouti showed mixed phylogeny with only two segregating sties and a near zero Tajima’s D score indicating no major selection. Out of 18 samples of crested lark, 13 birds from 4 different geographical regions showed identical sequences, while 3 birds from Russia and 1 from Cyprus grouped in a separate cluster and 1 crusted lark from Djibouti differed from all. The mean nucleotide difference and nucleotide diversity were 0.6993 and 0.0014, while a negative Tajima’s D of −1.1956 indicated the presence of rare alleles. Phylogenetic analysis of 9 different species of Saudi Arabian birds showed the discriminatory power of COI barcodes as all the different species grouped separately in specific clusters. ConclusionsCOI barcodes are not only indispensable tools for species identification but can also be used for analyzing molecular diversity and phylogenetic inference.

Genetic characterization of fish, Cynoglossus lingua Hamilton, 1822 found in Chandipur Coast of Odisha using COI and Cytochrome b genes

Aim: In this study we characterized the fish Cynoglossus lingua collected from Chandipur Coast of Odisha using the mtDNA markers cytochrome c oxidase I (COI) and cytochrome b (Cyt-b) genes. Methodology: Samples of flat fish were collected from Balaramgadi fish landing center, Chandipur sea beach, Odisha coast. The collected specimens were initially identified based on the morphology using standard taxonomic keys. Molecular characterization was accomplished through PCR amplification of COI and Cyt-b genes. The sequences were subjected to BLAST analysis and aligned using Clustal W for homology mapping. Phylogenetic status was inferred by the neighbor-joining method using MEGA v7.0. Results: The taxonomic keys and the COI and Cyt-b gene sequences identified the specimens as C. lingua. The COI and Cyt-b gene sequences showed 98% and 99% sequence similarity with C. lingua isolate FMU.CIFRI.FF-7 COI gene (GenBank accession: MN889532.1) and the C. lingua isolate FM.CIFRI.CYTB.FF-7 Cyt-b gene (GenBank accession MT362541.1), respectively. Interpretation: The study supports the reliability of combining classical taxonomic approaches with molecular analysis using COI and Cyt-b genes for the identification of C. lingua. Key words: Cynoglossus lingua, COI gene, Cytochrome b gene, DNA barcoding, Molecular taxonomy

Genetic diversity and phylogenetic analysis of mayfly Caenis (Insecta: Ephemeroptera) using Cytochrome C Oxidase I (COI) and 12s rRNA genes from Thailand

Abstract. Prakrongrak N, Boonsoong B, Monthatong M. 2023. Genetic diversity and phylogenetic analysis of mayfly Caenis (Insecta: Ephemeroptera) using Cytochrome C Oxidase I (COI) and 12s rRNA genes from Thailand. Biodiversitas 24: 1989-1997. Mayflies in genus Caenisis one of the top fifth species richnessbelonged to Family Caenidae. The present study aimed to use the partial mitochondrial Cytochrome COxidase subunit I (COI) and 12s rRNA nucleotide sequences for molecular species identification, diversity and phylogenetic relationships of mayflies in genus Caenis Stephens, 1835 species from Thailand. From a total of 37 specimens, thirteen nymphs were morphologically identified into five species: Caenis nasuta Malzacher, C. Picea Kimmins, C. ulmeriana Malzacher, C. cornigera Kang and Yang and C. longiforcipata Malzacher. The other 24 samples were Caenis sp.1 to sp.5. Partial COI sequences of all samples were compared with BOLD and GenBank for species identification. However, the result showed only genus-level identification as Caenis with greater than 80% similarity. For species delimitation, interspecific genetic distance among these species ranged from 14.4 to 26.6% (COI) and from 7.36 to 22.4% (12s rRNA). Intraspecific divergence levels were 0.0% to 2.35%. ABGD analysis of both genes divided data into 10 groups corresponding to 10 morphospecies of Caenis. The phylogenetic relationships of Caenis, COI and 12s rRNA genes also classified each species to well supported clusters. In addition, we report C. cornigera from Thailand for the first time. The COI and 12s rRNA phylogenetic trees indicate a close relationship between C. cornigera and C. longiforcipata, which is supported by their similar morphology.

Towards understanding insect species introduction and establishment: A community-level barcoding approach using island beetles.

Since Darwin put forward his opposing hypotheses to explain the successful establishment of species in areas outside their native ranges, the preadaptation and competition-relatedness hypotheses, known as Darwin's naturalization conundrum, numerous studies have sought to understand the relative importance of each. Here, we take advantage of well-characterized beetle communities across laurel forests of the Canary Islands for a first evaluation of the relative support for Darwin's two hypotheses within arthropods. We generated a mitogenome backbone tree comprising nearly half of the beetle genera recorded within the Canary Islands for the phylogenetic placement of native and introduced species sampled in laurel forests, using cytochrome c oxidase I (COI) sequences. For comparative purposes, we also assembled and phylogenetically placed a data set of COI sequences for introduced beetle species that were not sampled within laurel forests. Our results suggest a stronger effect of species preadaptation over resource competition, while also revealing an underappreciated shortfall in arthropod biodiversity data-knowledge of species as being native or introduced. We name this the Humboldtean shortfall and suggest that similar studies using arthropods should incorporate DNA barcode sequencing to mitigate this problem.

Population genetics of freshwater stingray require investigation to confirm DNA contamination: Reply to Alshari et al. (2023)

Alshari et al. (2023; Endang Species Res 50:311-313) were able to collect 3 Fluvitrygon kittipongi specimens from the Pahang River, which were not available to us at the time of our study (Lim &amp; Then 2022; Endang Species Res 48:43-50). The cytochrome c oxidase I (COI) sequences (or haplotypes) of these 3 stingrays were identical to each other but differed from the haplotypes of our Perak River specimens. The result of 100% similarity between COI sequences of our Perak River rays and the eDNA water samples isolated from Pahang River in our study was suggested by Alshari et al. (2023) as DNA contamination in our study, which is plausible. However, further population genetics studies would be necessary to ascertain that the Perak River haplotype does not occur in stingrays of the Pahang River.

DNA contamination and taxonomic diversity hinder eDNA research on freshwater stingray species: Comment on Lim &amp; Then (2022)

Recently, Lim &amp; Then (2022; Endang Species Res 48:43-50) used an environmental DNA (eDNA) PCR-based method for detecting the presence of an endangered species of freshwater stingray, Fluvitrygon kittipongi (Dasyatidae), in the Pahang River basin, east Peninsular Malaysia. For that, they designed a species-specific pair of primers to amplify a 196 base pair (bp) fragment of the cytochrome c oxidase I (COI) gene. Lim &amp; Then (2022) reported positive detection of this species along an upstream stretch of the Pahang River during the inter-monsoon and the southwest monsoon seasons, with the PCR-amplified eDNA sequences identical to the reference COI sequences of F. kittipongi collected from the Perak River basin, west Peninsular Malaysia. Here, we argue that such positive results are likely the consequence of a DNA contamination because the 196 bp COI fragments of F. kittipongi from the Pahang River and the Perak River differ from each other by 4 nucleotide substitutions. The source of contamination in Lim &amp; Then (2022) could have been the Perak samples the authors handled to develop the primers. We also briefly discuss the potential impact of the presumed co-occurrence of freshwater stingrays of the genus Fluvitrygon in the Pahang River on the specificity of the F. kittipongi-specific primers designed by Lim &amp; Then (2022).

Evidence of the Intrusion of the Oceanic Lightfish (Vinciguerria nimbaria) into Korean Waters Based on High-Throughput Sequencing of Mixed Fish Eggs

The oceanic lightfish Vinciguerria nimbaria is a mesopelagic species found in tropical and subtropical waters. In this study, we collected a total of 266 mixed fish egg samples from 78 stations in Korean waters of the Northwest Pacific Ocean from January to November 2021, and analysed these samples for V. nimbaria using cytochrome c oxidase I (COI) metabarcoding. We detected V. nimbaria eggs five times in May and once in August, with 20 V. nimbaria eggs to be estimated among the 266 mixed samples, which consisted of 68,844 eggs. To verify the accuracy of the metabarcoding results, two samples consisting of 1 and 6 eggs (diameter, 0.82 ± 0.07 mm; n = 5), respectively, that were identified as V. nimbaria were reanalysed using partial COI regions with the Sanger sequencing method. COI sequences obtained using both sequencing methods showed 100% identity in the overlapping regions. The mixed eggs formed one clade with V. nimbaria (average pairwise genetic distance, 0.002 ± 0.003; n = 7) in a phylogenetic ML tree based on the mitogenome (2 rRNAs and 13 protein-coding genes) of order Stomiiformes, including partial COIs from the mixed egg samples. The pairwise genetic distances in this clade were smaller than that of Stomiiformes (0.468 ± 0.081), except for V. nimbaria. These eggs represent direct evidence of the intrusion or distribution of adult V. nimbaria, an unrecorded species, in Korean waters.

First Report of Crown and Root Rot Caused by Phytopythium helicoides on Photinia × fraseriDress in China.

Photinia × fraseriDress is a hybrid species of Rosaceae and Photinia genus which is widely cultivated in China.During 2020 and 2021, approximately 80% of plants growing in Xuanwu district of Nanjing, China, exhibited disease symptoms including blight, necrosis, and dieback of crowns and roots. Symptomatic root tissues collected from 2-year-old plants were rinsed with water, cut into 2-mm tissues which were surface-sterilized in 70% ethanol for 60 s, and plated onto 10% V8 PARP agar and incubated in the dark at 26°C for 3 days. Hyphae emerged from 70% of the samples. Two representative isolates (PF-he2, PF-he3) were obtained and deposited. Ten agar plugs (2×2 mm2) of each isolate were transferred into 10 mL of 10% V8 juice to produce mycelial mats. To stimulate sporangial production, 3-5 drops of soil extract solution (soil collected from healthy fields, immersed in sterile water, and filtered) were added to each plate. Sporangia were terminal, ovoid to globose or papillate. The zoospores were 7.1-9.3 µm in diameter. Each oogonium contained a single, smooth, spherical and aplerotic oospore with a diameter of 24.5-32.6 μm. These morphological properties resemble those of Phytopythium helicoides (CBS286.31 from S. F. Ashby). For molecular identification, the large subunit (LSU) rDNA, cytochrome c oxidase I (COXI) gene, and COXII gene were amplified using the primer pairs NL1/NL4 , FM55/FM52R , and FM66/FM58 . The LSU, COXI, and COXII sequences of isolate PF-he2 were 100% (763/763 nt), 98.07% (1066/1087 nt), 99.64% (561/563 nt) identical to isolate CBS 286.31 (AY598665.2), GDGJ6 (KT750956.1), and TC3 (MN952224.1), respectively. Based on the morphological and molecular analysis, the two isolates shared 100% homology were identified as Phytopythium helicoides. The pathogenicity of two isolates were tested on potted 2-yr-old (40-cm tall) P. × fraseri. Six plants were dug up to expose root balls which were wounded before inoculations with a sterile needle, and then inoculated with zoospore suspension (106 zoospore/mL). Controls were treated with ddH2O. Three seedlings/isolate were used for each treatment including controls. All plants were repotted using the original sterilized potting mix and pots. After inoculation, the plants were covered with plastic bags, and sterilized H2O was sprayed into the bags twice per day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25/16 °C. All the inoculated plants showed lesions similar to those observed in the field after 23 days , whereas controls were asymptomatic. The isolates were reisolated from the lesions and sequenced as P. helicoides which has found causing root rot on Nelumbo nucifera, Rhododendron pulchrum, Zea mays in China, and also on Fragaria × ananassa in America, Peach Rootstock in California. Globally, this is the first report of P. helicoides causing crown blight and root rot of P. × fraseri. Management programs are under development to contain the spread of P. helicoides and treat diseased plants.

Juvenile drift of an invasive crayfish Faxonius virilis (Hagen, 1870) (Decapoda: Astacidea: Cambaridae) in the middle Pecos River, New Mexico, USA

Abstract Ecologists have long recognized that dispersal capabilities influence range expansion by nonnative species. Some of the most disruptive crayfish invaders are highly mobile as adults within and across watershed networks. In this study, we contribute a new facet to our understanding of crayfish early life history and invasions by documenting strong evidence of synchronized, behavioral downstream drift by early juveniles. During 2019, we observed a relatively dense springtime pulse of juvenile crayfish in drift nets set at two sites in the middle Pecos River near Fort Sumner, New Mexico. To determine whether this was an anomalous episode or an annual event, we sampled in 2020 and 2021 to: 1) determine the timing and extent of drifting behavior relative to environmental variation, and 2) identify the drifters to species level using DNA barcoding. Across three years, we collected juvenile crayfish (2.4–13.1 mm carapace length) after sunset during April through June in relatively clear water conditions (5–64 NTU) that coincided with minimum flows (41–152 cfs) and seasonally typical temperatures for this region (15–26 °C). To identify specimens to species level, we sequenced the barcoding region of the cytochrome c oxidase I (COI) DNA barcode for 35 juveniles and 6 adult male vouchers that represented all years. Based on morphological identifications of adult males and COI sequence comparisons to published reference specimens, nearly all drifting juveniles were identified as Virile Crayfish Faxonius virilis (Hagen, 1870). Drifters matched the sizes of second-molt juveniles newly released in the laboratory from two brooding females that we captured in the field. Faxonius virilis has been collected throughout the Rio Grande watershed, but not yet been reported in the Pecos River. As drifting early life stages are common across crustaceans, we suspect juvenile crayfish drift is probably not unique to F. virilis nor to our study system. We encourage others to perform targeted surveys of juvenile drift across a broad range of study systems, and to identify causal factors and ecological consequences of juvenile drift using controlled experiments.

DNA barcoding as an approach for species traceability and labeling accuracy of fish fillet products in Thailand

The lack of regulations and serious enforcement in the traceability of fishery products in some countries is one cause of food fraud, especially species substitution. Thailand is one of those countries where regulations are not fully enforced. Therefore, the labeling situation needs to be investigated in fish fillet products. Fifty-four products were collected from several supermarkets in Thailand. The labeling and molecular identity of each product were assessed by the application of a DNA barcoding technique on a cytochrome c oxidase I (COI) gene fragment of approximately 650 bp. All samples were successfully identified at the species level with 98–100% similarity in both the GenBank and BOLD databases. Only one sample could be identified at the genus level because the highest COI sequence match in the database was reported only at the genus level. Among all products, there were 25 species and 18 genera. Only one species, Pangasianodon hypophthalmus , was assessed as an endangered species according to IUCN status. FishBase was utilized to investigate the scientific names from common or market names. Two criteria were used to determine mislabeling, including i) mismatches between the scientific name identified by DNA barcoding and product labels and ii) the absence of common or market names in FishBase. Eighteen samples (33.33%) were mislabeled, which included 16 samples (88.89%) and 2 samples (11.11%) for criteria 1 and 2, respectively. Only one (11.11%) of nine products labeled with scientific names was mislabeled. Therefore, labeling products with scientific names may be a tool to reduce mislabeling to protect consumer rights. • Fifty-four Thai fish fillet products were examined for their species traceability and labeling accuracy using DNA barcodes. • Eighteen products (33.33%) were mislabeled. • Atlantic salmon-labeled products were most often mislabeled. • Only nine products were labeled with scientific names, which were very accurate. • Promoting labeling with scientific names may be one tool to reduce food fraud.

DNA barcodes in identification of some species of hawkmoths (Lepidoptera: Sphingidae)

The use of DNA barcodes is a molecular method commonly used for the identification of specimens that are difficult to pinpoint accurately by traditional morphological methods. DNA barcoding uses standard short genomic regions that are universally present in target lineages and has sufficient sequence variation to identify species in the genus. The hawkmoth genus Cechetra (Zolotuhin &amp; Ryabov, 2012) was proposed for a group of morphologically similar, stripe-patterned species. Based on the seven specimens of the three species of Cechetra genus collected from the Central of Vietnam, we used morphological analysis to initially identified their scientific name of Cechetra lineosa (Walker, 1856) for 04 specimens (LB_F01, LB_G01, LB_H01 and LK_G05); 02 specimens (MK_H04 and MK_B07) were identified with the scientific name of Cechetra minor (Butler, 1875) and 01 specimen (SG_D7) with scientific name Cechetra subangustata (Rothschild, 1920). Then, the COI (Cytochrome C oxidase I) sequence was used for molecular analysis. Total DNA was extracted from the legs of specimens, COI with over 600 bp in length from each specimen was amplified by PCR reactions. The PCR products are hence purified and sequenced. COI gene cloning results showed a very high level of genetic similarity (over 99%) and 7 specimens are of the genus Cechetra, Sphingidae family. For three homomorphic species belonging to the genus Cechetra Zolotuhin &amp; Ryabov, 2012 including Cechetra lineosa (Walker, 1856); Cechetra minor (Butler, 1875) and Cechetra subangustata (Rothschild, 1920). The studied sequences have been added to the current database at GenBank (NCBI) with accession numbers MT994230, MT994231, MT994232, MT994233, MT994234, MT994235, and MT994236. 

Combining mitochondrial DNA and morphological data to delineate four new millipede species and provisional assignment to the genus

Hitherto, the millipede genus Apeuthes (family Pachybolidae, subfamily Trigoniulinae) was only known from three species described in Vietnam based on morphological characters. The present study uses two partial mitochondrial gene fragments (cytochrome c oxidase I (COI) and 16S ribosomal RNA) and morphology to define four new Apeuthes species from Malaysia, Thailand and Vietnam: A. fimbriatus, sp. nov., A. longeligulatus, sp. nov., A. pollex, sp. nov. and ?A. spininavis, sp. nov. The intraspecific COI sequence divergence of two Apeuthes species is 3–7% (mean: 5%) and the interspecific divergence of five species is 11–16% (mean: 13.7%). All members of the genus share unique male characters, viz the posterior gonopod telopodite with several dentate, serrate or tuberculate lamellae in a boat-like cavity or a boat-like cavity covered with spines. The delimitation of the four new species is supported by the congruence between mitochondrial DNA and morphological data. However, while the monophyly of Trigoniulinae is well supported, the relationships within this subfamily, and particularly among Apeuthes species, including the monophyly of Apeuthes, lack strong support. Therefore assignment of the four new species, and particularly of ?A. spininavis sp. nov., to the genus Apeuthes is tentative and awaits a comprehensive revision of the group.

Characterization of spiny lobsters from Bangladesh waters using morphology, COI and 16S rRNA sequences

This study aims to taxonomically identify and characterise the phylogenetic relationships of spiny lobsters based on mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes from Bangladesh waters. A total of 19 barcode sequences (10 partial COI sequences and 9 partial 16S rRNA) were successfully generated from 12 collected spiny lobster samples representing four species belonging to the family Palinuridae. The average genetic distances within and between species were 0.834 ± 0.427 and 17.810 ± 0.830, respectively, in COI and 0.107 ± 0.255 and 8.401 ± 2.547, respectively, in 16S rRNA genes. The successful amplification rate of 16S rRNA was higher than that of the COI marker. In the maximum likelihood (ML) tree, the sequences of the same species were clustered together under a single clade for both COI and 16S rRNA, which supports the efficacy of both marker genes in differentiating lobster species.