Identification of CITES-Listed Euphorbia royleana through DNA Barcoding Technology: A New Facet in Wildlife Forensics

Disorganized and chaotic collection of the Euphorbia plant species from the wild is one of the major reasons for its endangered status. According to CITES, the trade in Euphorbia royleana species is prohibited under Appendix II. However, the trade continues unabated as current identification methods do not discriminate between closely related species. In the present study, a DNA barcoding method has been used to establish inter- and intra-specific divergences of both matK and rbcL regions by using pairwise genetic distance measurement methods for evaluating the maximum barcoding gap. The matk and rbcL yielded a 100% amplification and sequencing success rate to distinguish closely related species of Euphorbia royleana unambiguously. The matk and rbcL showed average interspecific genetic distance divergence values of 0.031and 0.015, respectively. The maximum number of species-specific SNPs was observed in matK sequences at seven consecutive sites, which could distinguish Euphorbia royleana from closely related species. The best candidate barcoding region to identify Euphorbia royleana was found to be matK with a single-locus barcoding approach. Furthermore, the species discrimination method was developed with the help of species-specific SNPs derived from the matK barcoding region to accurately authenticate Euphorbia royleana, and it provided 100% species resolution

Illuminating species diversity of nudibranch in Indonesian coral reef ecosystem using molecular identification

Nudibranch has high species diversity with complex morphological characters and is challenging to identify at the species level. The lack of knowledge about nudibranchs makes it difficult to identify conventionally using morphological characters. This study aims to identify nudibranchs at the species level using the DNA barcoding method from the mitochondrial cytochrome oxidase 1 (CO1) gen. The results of DNA barcoding using the Cytochrome Oxidase I (COI) gene showed 18 species of 51 samples analyzed. The phylogenetic tree reconstruction revealed 11 main clades belonging to 11 genera. The genetic distance between and within species clearly shows the difference between individuals. Interspecific genetic distance shows the lowest value between species was found between Chromodoris annae and Chromodoris magnifica is 0.075, and the largest genetic distance observed between species Glossodoris rufomarginata and Tritonidae sp is 0.354. This study shows molecular analysis can be used to identify nudibranch up to species level, which will be a source of information in knowing the distribution and the genetic distance.

Adding pieces to the puzzle: insights into diversity and distribution patterns of Cumacea (Crustacea: Peracarida) from the deep North Atlantic to the Arctic Ocean

The Nordic Seas have one of the highest water-mass diversities in the world, yet large knowledge gaps exist in biodiversity structure and biogeographical distribution patterns of the deep macrobenthic fauna. This study focuses on the marine bottom-dwelling peracarid crustacean taxon Cumacea from northern waters, using a combined approach of morphological and molecular techniques to present one of the first insights into genetic variability of this taxon. In total, 947 specimens were assigned to 77 morphologically differing species, representing all seven known families from the North Atlantic. A total of 131 specimens were studied genetically (16S rRNA) and divided into 53 putative species by species delimitation methods (GMYC and ABGD). In most cases, morphological and molecular-genetic delimitation was fully congruent, highlighting the overall success and high quality of both approaches. Differences were due to eight instances resulting in either ecologically driven morphological diversification of species or morphologically cryptic species, uncovering hidden diversity. An interspecific genetic distance of at least 8% was observed with a clear barcoding gap for molecular delimitation of cumacean species. Combining these findings with data from public databases and specimens collected during different international expeditions revealed a change in the composition of taxa from a Northern Atlantic-boreal to an Arctic community. The Greenland-Iceland-Scotland-Ridge (GIS-Ridge) acts as a geographical barrier and/or predominate water masses correspond well with cumacean taxa dominance. A closer investigation on species level revealed occurrences across multiple ecoregions or patchy distributions within defined ecoregions.

COI mtDNA barcoding and morphology for the description of a new species of ricinuleid of the genus Pseudocellus (Arachnida: Ricinulei: Ricinoididae) from El Triunfo Biosphere Reserve, Chiapas, Mexico

A new species of epigean ricinuleid of the genus Pseudocellus Platnick, 1980 from El Triunfo Biosphere Reserve, Chiapas, Mexico is described. DNA barcoding utilizing mitochondrial cytochrome c oxidase subunit 1 (CO1) and morphology were used for species delimitation. Molecular analyses and species delimitation included four methods: 1) General Mixed Yule Coalescent model (GMYC), 2) Automatic Barcode Gap Discovery (ABGD), 3) Bayesian Poisson Tree Process (bPTP), and 4) Assemble Species by Automatic Partitioning (ASAP). All molecular methods and morphology were consistent in delimiting and recognizing the new species described herein. The average interspecific genetic distance (p-distance) among analyzed species of Pseudocellus was 11.6%. The species is described based on adult males and females: Pseudocellus giribeti sp. nov. This is the seventh species described from Chiapas, which holds the highest number of ricinuleids species for the country. The total number of described species of Pseudocellus from Mexico increases to 21, having the highest species diversity of known ricinuleids worldwide.

Revision of Simulium rufibasis (Diptera: Simuliidae) in Japan and Korea: Chromosomes, DNA, and Morphology

The widespread nominal black fly Simulium (Simulium) rufibasis Brunetti was reexamined morphologically, chromosomally, and molecularly to determine the status of populations in Japan and Korea with respect to S. rufibasis from the type locality in India and to all other known species in the S. (S.) tuberosum species-group. Morphological comparisons established that the species previously known as S. rufibasis in Japan and Korea is distinct from all other species. Consequently, it was described and illustrated as a new species, Simulium (S.) yamatoense. Simulium yokotense Shiraki, formerly a synonym of S. rufibasis, was morphologically reevaluated and considered a species unplaced to species-group in the subgenus Simulium. Chromosomal analyses of S. yamatoense sp. nov. demonstrated that it is unique among all cytologically known species of the S. tuberosum group and is the sister species of the Taiwanese species tentatively known as S. (S.) arisanum Shiraki. Populations of S. yamatoense sp. nov. included two cytoforms, based on the sex chromosomes. Cytoform A, including topotypical representatives, was found in Kyushu, Japan, whereas cytoform B was found in Korea and Honshu, Japan. Molecular analysis based on the COI mitochondrial gene generally corroborated morphological and chromosomal data that S. yamatoense sp. nov. is a distinct species and, like the chromosomal data, indicate that it is most closely related to S. arisanum, with interspecific genetic distance of 2.92–4.63%.

Comparison of the Complete Eragrostis pilosa Chloroplast Genome with Its Relatives in Eragrostideae (Chloridoideae; Poaceae)

Eragrostis of the tribe Eragrostideae is a taxonomically complex genus, because of its polyploid nature and the presence of similar morphological characters among its species. However, the relationship between these morphologically indistinguishable species at the genomic level has not yet been investigated. Here, we report the complete chloroplast genome of E. pilosa and compare its genome structures, gene contents, simple sequence repeats (SSRs), sequence divergence, codon usage bias, and Kimura 2-parameter (K2P) interspecific genetic distances with those of other Eragrostideae species. The E. pilosa chloroplast genome was 134,815 bp in length and contained 132 genes and four regions, including a large single-copy region (80,100 bp), a small single-copy region (12,661 bp), and a pair of inverted repeats (21,027 bp). The average nucleotide diversity between E. pilosa and E. tef was estimated to be 0.011, and 0.01689 among all species. The minimum and maximum K2P interspecific genetic distance values were identified in psaA (0.007) and matK (0.029), respectively. Of 45 SSRs, eight were shared with E. tef, all of which were in the LSC region. Phylogenetic analysis resolved the monophyly of the sampled Eragrostis species and confirmed the close relationship between E. pilosa and E. tef. This study provides useful chlorophyll genomic information for further species identification and phylogenetic reconstruction of Eragrostis species.

Integrating DNA Barcoding and Traditional Taxonomy for the Identification of Dipterocarps in Remnant Lowland Forests of Sumatra

DNA barcoding has been used as a universal tool for phylogenetic inferences and diversity assessments, especially in poorly studied species and regions. The aim of this study was to contrast morphological taxonomy and DNA barcoding, using the three frequently used markers matK, rbcL, and trnL-F, to assess the efficiency of DNA barcoding in the identification of dipterocarps in Sumatra, Indonesia. The chloroplast gene matK was the most polymorphic among these three markers with an average interspecific genetic distance of 0.020. The results of the molecular data were mostly in agreement with the morphological identification for the clades of Anthoshorea, Hopea, Richetia, Parashorea, and Anisoptera, nonetheless these markers were inefficient to resolve the relationships within the Rubroshorea group. The maximum likelihood and Bayesian inference phylogenies identified Shorea as a paraphyletic genus, Anthoshorea appeared as sister to Hopea, and Richetia was sister to Parashorea. A better discriminatory power among dipterocarp species provided by matK and observed in our study suggests that this marker has a higher evolutionary rate than the other two markers tested. However, a combination of several different barcoding markers is essential for reliable identification of the species at a lower taxonomic level.

New records and their associated DNA barcodes of the butterfly family Hesperiidae in Tibet, China

The specimens of the family Hesperiidae collected from Tibet during 2016–2018 are identified using morphology. COI sequences of 76 individuals are newly obtained. The result of our morphological study is congruent with COI gene analyses. Maximum likehood (ML) and Bayesina inferences (BI) analyses reveal that individuals identified morphologically as the same species cluster cohesively. The minimum interspecific genetic distance is 1.7% between Halpe aucma and H. filda, and the genetic distance between conspecific individuals ranged from 0 to 0.2% for the genus Halpe. A total of 51 species are recognized, and six of them, Celaenorrhinus consanguineus Leech, 1891, Barca bicolor (Oberthür, 1896), Aeromachus propinquus Alphéraky, 1897, Pedesta bivitta (Oberthür, 1886), Baoris penicillata chapmani Evans, 1937, and Ochlodes brahma Moore, 1878, are reported from Tibet for the first time, and the last species is new to China.

A new species of black fly (Diptera: Simuliidae) in the Simulium (Simulium) multistriatum species-group from Thailand

A new black fly species of the Simulium multistriatum species-group of the subgenus Simulium Latreille is described from the mountainous area in northeastern Thailand, based on morphology and mitochondrial DNA sequences. The new species is morphologically similar to S. laui Takaoka and Sofian-Azirun and S. lacduongense Takaoka and Ya’cob originally described from Vietnam, S. fenestratum Edwards originally described from Indonesia and S. chaliowae Takaoka and Boonkemtong originally described from Thailand, but can be distinguished in the adult stage by the number of upper eye facets and globular shape of the spermatheca and in the pupal stage by the cocoon and shape of thoracic tubercles. Genetic distance and phylogenetic analyses of mitochondrial cytochrome c oxidase I sequences differentiated the new species from other members of S. multistriatum species-group. All specimens of the new species formed a monophyletic clade with strong support in all phylogenetic analyses. The minimum interspecific genetic distance of 4.9% is considerably greater than the new species maximum intraspecific genetic distance (2.7%).

COI barcoding of plant bugs (Insecta: Hemiptera: Miridae)

The family Miridae is the most diverse and one of the most economically important groups in Heteroptera. However, identification of mirid species on the basis of morphology is difficult and time-consuming. In the present study, we evaluated the effectiveness of COI barcoding for 123 species of plant bugs in seven subfamilies. With the exception of threeApolygusspecies—A. lucorum,A. spinolae, andA. watajii(subfamily Mirinae)—each of the investigated species possessed a unique COI sequence. The average minimum interspecific genetic distance of congeners was approximately 37 times higher than the average maximum intraspecific genetic distance, indicating a significant barcoding gap. Despite having distinct morphological characters,A. lucorum,A. spinolae, andA. watajiimixed and clustered together, suggesting taxonomic revision. Our findings indicate that COI barcoding represents a valuable identification tool for Miridae and can be economically viable in a variety of scientific research fields.