Reliable species identification is critical for detection and monitoring of biological invasions. In this study, we propose four sets of primers for efficient amplification of several loci, including the mitochondrial cytochrome oxidase-c (COI) subunit I gene which is a basis for DNA barcoding. This set of primers gives a shorter product which can be used in high-throughput sequencing systems for metabarcoding purposes. Another mitochondrial locus encoding the large ribosomal subunit (16S) may be useful to study the population structure and as an additional source of information in the metabarcoding of communities. We propose to use a set of primers for the nuclear locus of the small ribosomal subunit (18S) as a positive control and to verify the results of the barcoding. Our proposed sets of primers demonstrate a high amplification efficiency and a high specificity both for freshwater alien and indigenous fishes. The proposed research design makes it possible to carry out extremely cheap studies on the assessment of biological diversity using genetic analysis without expensive equipment, and with the technique for conducting laboratory work and processing of the results available to any researcher. The paper also presents original data on the genetic polymorphism of all mass alien fish species in the Volga-Kama region. High efficiency of DNA identification based on our primers is shown as compared to traditional monitoring of biological invasions.

We aimed to apply DNA barcoding tool for the molecular identification of horn snails T. telescopium using mitochondrial cytochrome oxidase I gene (mt-COI) and to investigate their evolutionary relationship along with location-specific bio-geographical variations. The molecular data sets of this study indicate that strong probability of T. telescopium species taxonomic confirmation using mt-COI sequences. Results of the phylogenetic analysis suggest that Telescopium sp. was monophyletic with disseminated nodes and the evolution of group II originated from group I. The substantial genetic distance among the mt-COI sequences (0.005 to 0.184) were noticed. Large divergence between the south-west coast of India and Australia region population indicates limited gene flow between the two continents. Our study suggests that the genera Telescopium is globally ubiquitous but genetically showing inter-region differentiation. We conclude that mt-COI gene can be used to identify gastropod T. telescopium species.

The purpose of this study was to identify candidate novel species performing beneficial ecological functions from soil. The hypothesis was that novel culturable species with antibiotic production potential could be readily found from soil in the Nature Canyon at Los Angeles Pierce College. 137 bacterial isolates from soil were grown on nutrient agar, ISP-6 medium, or LB agar and partial 16S was ascertained using the Sanger platform for regions V1-V4. One of the potentially novel isolates, Pseudomonas sp. 21-C3-ER, was isolated on nutrient agar from fallow soil in an agricultural nature preserve in Los Angeles. The soil conditions were 3.7% moisture, clay texture by touch, 13.37% organic matter, pH=6.63, TDS=638, 150 ppm nitrogen, 114 ppm phosphate, and 42 ppm potash. Whole genome sequencing was performed on the DNBseq platform and species identification was performed with TRUEBacID; the closest relative was Pseudomonas brassicacearum subsp. neoaurantiaca which was 92.05% similar by genomic evidence. 197 virulence factors were identified including flagellar biosynthesis, motor proteins, Beta-lactamase, Fosfomycin, copper, and sulfur drug resistance. Gram stain of strain 21-C3-ER revealed gram negative rods. Rods were visualized with scanning electron microscopy, revealing individuals approximately 1 µm in length. Secondary metabolite analysis using antiSMASH showed a variety of post-translationally modified proteins and a promysalin analog. The qualitative test for pyoverdine siderophore production using UV light was positive. Metabolic analysis using RAST indicated genes were present for pyoverdine production, antimicrobial resistance, and ammonification. The nitrate reduction test was positive. Denitrifying bacteria may prevent leaching of nitrates from soil, since ammonia has a higher affinity to soil and organic matter than nitrate. We propose Pseudomonas fernandeno as a novel species. The type stain is 21-C3-ER.

Barcoding of DNA is a species identification technique that utilizes a small DNA fragment from a specific gene or genes known as "sequencing." The idea behind DNA barcoding is that a single sequence can be used to specifically identify an organism to species by comparison with a reference library of these DNA sections. Herbal adulteration can be monitored using DNA barcodes that utilize medicinal plants such as Atropa belladonna. Because it is challenging to distinguish between species solely based on morphology, other plants belonging to the same genus are replacing it in trade. Such replacement with plants not utilized for medical purposes could have negative side effects. Also, Atropa belladonna's positive effects can be completely lost. Taxonomists also have trouble distinguishing the genuine Atropa belladonna plant from cultivated items. As a result, DNA barcoding can now be used to overcome this challenge. The development of DNA barcodes may help with trade control, which protects and conserves endangered species from export outside of the country due to their economic and biological significance. In this review, we discuss the DNA barcoding of Atropa belladonna plant in the medicinal plants field.

French Guiana, located in the Guiana Shield, is a natural reservoir for many zoonotic pathogens that are of considerable medical or veterinary importance. Until now, there are few data available on the description of parasites circulating in this area, especially on protozoan belonging to the phy-lum Apicomplexa; conversely, the neighbouring countries describe a high parasitic prevalence in animals and humans. Epidemiological surveillance is necessary, as new potentially virulent strains may emerge from these forest ecosystems, such as Amazonian toxoplasmosis. However, there is no standard tool for detecting protozoa in wildlife. In this study, we developed Meat-Borne-Parasite, a high-throughput meta-barcoding workflow for detecting Apicomplexa based on Oxford Nanopore Technologies sequencing platform using the 18S gene of fourteen Apicomplexa-positive samples collected in French Guiana. Sequencing reads were then analysed with MetONTIIME pipeline. Thanks to a scoring rule, we were able to classify as Apicom-plexa-positive 10 samples out of 14 and reveal the presence of co-carriages. The same samples were also sequenced with Illumina platform, for validation purposes. On samples identified as Apicomplexa-positive by both platforms, a strong positive correlation at up to genus level was reported. Overall, the presented workflow represents a reliable method for Apicomplexa detec-tion, which may pave the way for more comprehensive biomonitoring of zoonotic pathogens.

In the last two decades, plant taxonomy has bloomed, following the development of a novel technique, namely DNA Barcoding. DNA barcodes are standardized sequences, ideally unique, coding or non-coding, either from the genome of the organism or from its organelles, that are used to identify/classify an organismal group; in short the method includes amplification of the DNA barcode, sequencing and comparison with a reference database containing the relevant sequences from different species. In plants, the use a universal DNA barcode, such as COI which is used in animals, has not been achieved so far. Several DNA barcodes from single loci as well as combinations of barcodes have been successfully used in various settings; additionally, as next generation sequencing becomes affordable, very long sequences- super barcodes, such as the chloroplast genome are being used successfully especially to distinguish between close species. In this review we summarize the work done on DNA barcoding in plants and discuss different approaches used so far.

The need for multi-gene analysis in evolutionary and taxonomic studies is generally accepted. However, sequencing of multiple genes is not always possible. For various reasons, short mito-chondrial DNA barcodes are the only source of molecular information for some species in many genera, although multilocus data are available for other species of the same genera. In particular, such a situation exists in the species-rich butterfly subgenus Polyommatus (Agrodiaetus). Here, we analysed partitioning of this subgenus into species groups by using three data sets. The first data set was represented by short mitochondrial DNA barcodes for all analysed samples. The second and third data sets were represented by combination of short mitochondrial DNA barcodes for a part of the taxa with longer mitochondrial sequences COI+tRNA-Leu+COII (data set 2) and with longer mitochondrial COI+tRNA-Leu+COII and nuclear 5.8S rDNA+ITS2+28S rDNA sequences (data set 3) for the remaining species. We show that DNA barcoding approach (data set 1) failed to reveal the taxonomic structure resulting in numerous polytomies in the phylogenetic tree ob-tained. Combined analysis of the mitochondrial and nuclear sequences (data sets 2 and 3) re-vealed the species groups and position within these species groups even for the taxa for which only short DNA barcodes were available.

Free-living terrestrial mites (Acari) have persisted through numerous glacial cycles in Antarctica. Very little is known, however, of their genetic diversity and distribution, particularly within the Ross Sea region. To redress this gap, we sampled for mites throughout the Ross Sea region, East Antarctica, including Victoria Land and the Queen Maud Mountains (QMM), covering a latitudinal range of 72-85oS, as well as from Lauft Island near Mt Siple (73oS) in West Antarctica and Macquarie Island (54oS) in the sub-Antarctic. We assessed genetic diversity using mitochondrial cytochrome c oxidase subunit I gene sequences (COI-5P DNA barcode region), and also morphologically identified voucher specimens. We obtained 130 sequences representing four genera: Nanorchestes (n = 30 sequences), Stereotydeus (n = 46), Coccorhagidia (n = 18) and Eupodes (n = 36). Tree-based analyses (maximum likelihood) revealed 13 genetic clusters, representing as many as 23 putative species indicated by Barcode Index Numbers (BINs) from the Barcode of Life Datasystems (BOLD) database. We found evidence for geographically-isolated cryptic species, e.g. within Stereotydeus belli and S. punctatus, as well as unique genetic groups occurring in sympatry (e.g. Nanorchestes spp. in QMM). Collectively, these data confirm high genetic divergence as a consequence of geographic isolation over evolutionary timescales. From a conservation perspective, additional targeted sampling of understudied areas in the Ross Sea region should be prioritised, as further diversity is likely to be found for these short-range endemic mites.

Systematics plays the most crucial role in biodiversity conservation which is at stake due to anthropogenic activities and environmental degradations. The ever-increasing decline of classical taxonomic expertise drives the need to develop molecular marker-based tools for quick, efficient, and reliable detection of organisms, to assess their ecological impacts for deepening our understanding of systematic and evolutionary relationships between organisms which is central to biology. The pace of alpha taxonomy has quickened by its integration with an increasingly fashionable and novel concept called DNA barcoding which utilizes a short genetic marker or barcode to categorize species for enhanced biodiversity assessment. As a supplementary but not complete alternative of systematics research, DNA barcoding, however, not error-free, brings precision in identification by solving existing problems of classical taxonomy and phylogenetics, irrespective of the growth stage of organisms, particularly for known taxa rather than unknown ones. Mitochondrial gene Cytochrome C Oxidase 1 (COI) serves as a universal animal barcode but there is no such universal barcode for plants and developing a suitable one is more challenging. With the recent advancement of Next Generation Sequencing (NGS), DNA metabarcoding technology is advancing rapidly. Still, ambiguity and error prevail with the correct identification of species due to some problems. After extensive analysis of the existing DNA barcoding papers, this review discusses commonly used DNA barcodes in plants and animals, their roles, advantages, limitations to solve existing problems of conservation biology and add the author’s views and recommendations.

Uncontrolled transmission of Mycobacterium tuberculosis (M. tuberculosis, MTB) drug resistant strains is a challenge to control efforts of global tuberculosis programme. Due to increasing multi-drug resistant (MDR) cases in Arunachal Pradesh, a northeastern state of India, the tracking and tracing of these resistant MTB strains is crucial for infection control and spread of drug resistance. This study aims to correlate the phenotypic DST, genomic DST (gDST) and phylogenetic analysis of MDR-MTB strains in the region. Of total 200 suspected MDR-MTB isolates, 125(62.5%) were identified as MTB. MGIT-960 SIRE DST detected 71/125(56.8%) isolates as MDR/RR-MTB of which 22(30.9%) were detected resistant to second line drugs. Whole genome sequencing of 65 isolates and their gDST found Ser315Thr mutation in katG (35/45;77.8%) and Ser531Leu mutation in rpoB (21/41;51.2%) associated with drug resistance. SNP barcoding categorized the dataset with Lineage2 (41;63.1%) being predominant followed by Lineage3 (10;15.4%), Lineage1 (8;12.3%) and Lineage4 (6;9.2%) respectively. Phylogenetic assignment by cgMLST gave insights of two Beijing sub-lineages viz; 2.2.1 (SNP difference < 19) and 2.2.1.2 (SNP difference < 9) associated with recent ongoing transmission in Arunachal Pradesh. This study provides first insight in identifying the ongoing transmission of two virulent Beijing sub-lineages associated with TB drug resistance.

Determining the DNA sequencing of a small element in the mitochondrial DNA (DNA barcoding) makes it possible to easily identify individuals of different larval stages of marine crustaceans without the need for laboratory rearing. It can also be used to construct taxonomic trees, although it is not yet clear to what extent this barcode-based taxonomy reflects more traditional morphological or molecular taxonomy. Collections of zooplankton were made using conventional plankton nets in Newport Bay and the Pacific Ocean near Newport Beach, California (Lat. 33.628342, Long. -117.927933) between May 2013 and January 2020, and individual crustacean specimens were documented by videomicroscopy. Adult crustaceans were collected from solid substrates in the same areas. Specimens were preserved in ethanol and sent to the Canadian Centre for DNA Barcoding at the University of Guelph, Ontario, Canada for sequencing of the COI DNA barcode. From 1042 specimens, 544 COI sequences were obtained falling into 199 Barcode Identification Numbers (BINs), of which 76 correspond to recognized species. The results show the utility of DNA barcoding for matching life-cycle stages as well as for documenting the diversity of this group of organisms.

In Zambia wild edible terrestrial orchids are used to produce a local delicacy called chikanda, which has become increasingly popular throughout the country. Commercialization puts orchid populations in Zambia and neighbouring countries at risk of overharvesting. Hitherto, no study has documented which orchid species are traded on local markets, as orchid tubers are difficult to identify morphologically. In this study, the core land-plant DNA barcoding markers rbcL and matK were used in combination with nrITS to determine which species were sold on Zambian markets. Eighty-two interviews were conducted to determine harvesting areas, as well as possible sustainability concerns. By using nrITS DNA barcoding, a total of 16 orchid species in six different genera could be identified. Both rbcL and matK proved suitable to identify the tubers up to genus- or family level. Disa robusta, Platycoryne crocea and Satyrium buchananii were identified most frequently and three previously undocumented species were encountered on the market. Few orchid species are currently listed on the global IUCN Red List. Local orchid populations and endemic species could be at risk of overharvesting due to the intensive and indiscriminate harvesting of chikanda orchids and we therefore encourage increased conservation assessment of terrestrial African orchids.

The genus Colias Fabricius, 1807 includes numerous taxa and forms with uncertain status and taxonomic position. Among such taxa are Colias mongola Alphéraky, 1897 and Colias tamerlana Staudinger, 1897, interpreted in the literature either as conspecific forms, as subspecies of different but morphologically somewhat similar Colias species, or as distinct species-level taxa. Based on mitochondrial and nuclear DNA markers, we reconstruct a phylogeographic pattern of the taxa in question. We recover and include in our analysis DNA barcodes of the century-old type specimens, the lectotype of C. tamerlana deposited in the Natural History Museum (Museum für Naturkunde), Berlin, Germany (ZMHU), the paralectotype of C. tamerlana and the lectoptype of C. mongola deposited in the Zoological Institute, Russian Academy of Sciences, Saint-Petersburg, Russia (ZISP). Our analysis grouped all specimens within four (HP_I-HP-IV) deeply divergent but geographically poorly structured clades which did not support nonconspecifity of C. mongola–C. tamerlana. We also show that all studied females of the widely distributed haplogroup HP_II were infected with a single Wolbachia strain belonging to the supergroup B, while the males of this haplogroup, as well as all other investigated specimens, were not infected. Our data underline the relevance of large-scale sampling dataset analysis and the need of testing for Wolbachia infection to avoid erroneous phylogenetic reconstructions and species misidentification.

The life cycles and biodiversity of Pacific coast gastropods were analyzed by videomicroscopy and DNA barcoding of indi-viduals collected from tide pools and in plankton nets from a variety of shore stations. In many species (Families Calyptrae-idae, Cerithiopsidae, Strombidae, Vermetidae, Columbellidae, Nassariidae, Olivellidae, Hermaeidae, Onchidorididae, Gas-tropteridae, Haminoeidae), the free-swimming veligers were recovered from plankton collections; in Roperia poulsoni (family Muricidae) veligers were usually recovered from egg sacs where they had been retained although some escapees were found in plankton collections; in Pteropurpura festiva (family Muricidae) free-living veligers were also found; and in Atlanta californiensis (family Atlantidae) both veligers and adults were obtained from plankton collections making this a holoplank-tonic species. The results confirm that DNA barcoding using the COI barcode is a useful strategy to match life-cycle stages within species as well as to identify species and to document the level of biodiversity within the gastropods.

Asian giant hornet, Vespa mandarinia, is an invasive species that could potentially destroy local honeybee industry in North America. It has been observed to nest in the coastal regions of British Columbia in Canada and Washington State in USA. What is the source population of the immigrant hornets? The identification of the source population can not only shed light on the route of immigration, but also on the similarity between the native habitat and the potential new habitat in the Pacific Northwest. We analyzed mitochondrial COX1 sequences of specimens sampled from multiple populations in China, South Korea, Japan and the Russian Far East. V. mandarinia exhibits phylogeographic patterns, forming monophyletic clades for 16 specimens from China, six specimens from South Korea and two specimens from Japan. The two mitochondrial COX1 sequences from Nanaimo, British Columbia are identical to the two sequences from Japan. The COX1 sequence from Blaine, Washington State, clustered with those from South Korea, and is identical to one sequence from South Korea (GenBank accession MN716828). Our geophylogeny, which allows visualization of genetic variation over time and space, provides evolutionary insights on the evolution and speciation of three closely related vespine species (V. tropica, V. soror and V. mandarinia). The geophylogeny also highlights a strong insufficiency in sample collection in China. The existing sequenced specimens from mainland China with geographic coordinates were represent only four provinces, missing populations along the coastal regions in East China. One therefore cannot exclude the possibility that the Asian giant hornet found in Canada and USA may also be identical to V. mandarinia in Eastern China. We strongly recommend more extensive DNA barcoding data with geographic information and longer DNA barcode (e.g., the entire mitochondrial genome) so that critical questions on invasive species and environmental conservation can be addressed more accurately than what is possible with existing data.

Infections with symbiotic single-celled organisms classified as Ciliophora, are commonly reported in various species of invertebrates. Freshwater mussels also play the role of a host for them, being responsible for their biodiversity. Knowledge about these relationships is insufficient. Freshwater mussels are among the most endangered groups of animals. Ciliate endosymbionts in mussels may be responsible for the diseases and increasingly frequent cases of mass mussel deaths and are particularly dangerous for commercially farmed mussels and for restocking of mussels as part of active conservation measures. On the other hand the Ciliophora parasites and commensals living in freshwater mussels is undescribed. Many of these species are likely to become extinct before they are known to science. Recently, two genera of Ciliophora, Conchophthirus sp. and Trichodina sp., have been described in the mantle cavity of U. crassus - an endangered and protected species in the EU. The basis of extensive research on the impact of endosymbiotic ciliates on U. crassus populations is the knowledge of their species composition, proportion of infected individuals, level of infection intensity and distribution in different types of rivers. Such studies were carried out in the mantle cavity of U. crassus from three rivers in three seasons. Cloning, sequencing and functional analysis of the genetic material of Ciliophora was carried out using NGS (Next-Generation Sequencing) analysis based on the hypervariable V4 and V9 regions of the 18SrRNA gene, which enables the identification of taxonomic groups, including genera and species. The most numerous OTUs are common and cosmopolitan species. But some commensals and potentially parasites were found too. Ciliophora associated with mantle cavity form an interesting, hierarchical biocomplex but their interactions with mussels need further studies.

To fill in the blanks for charophytes in the Caucasus to improve our knowledge of species distribution areas in Eurasia, a field survey and study of available specimens from Dagestan (North Caucasus, Russia) was conducted based on morphological observation and molecular genetic analysis. Seventeen new localities for six Chara species and one Tolypella species, with six new species and one new genus for the region studied, and one new species for the Caspian Sea region were found. Some species records changed the outline or filled in the gaps in the species distribution area. The presence of species with a distribution area mainly in the centre of Eurasia (C. globata, C. neglecta) with mainly Mediterranean – Middle East species (C. gymnophylla) is notable for this region, as well as for other studied regions of the Caucasus having a combination of species with different distribution areas. The frequent occurrence of C. gymnophylla points is similar to that in the Mediterranean region and the Middle East. Small brackish waterbodies at the coast of the Caspian Sea, freshwater mountain rivers, small waterbodies associated with them, and water reservoirs are the main habitats of charophytes in the regions. The habitat preference and distribution in the Caucasus allowed suggestion some species for protection. The absence of endemic species among charophytes from Dagestan and Caucasus contrasts with the flora of terrestrial magnoliophytes, which have a large number of endemic species.

Ophiura sarsii is a common brittle star species across Arctic and subarctic regions of Atlantic and Pacific oceans. In the Barents Sea O. sarsii is among the dominant echinoderms. We studied genetic diversity of O. sarsii by sequencing the 548 bp fragment of mitochondrial COI gene. O.sarsii demonstrated high genetic diversity in the Barents Sea. Both major Atlantic mtDNA lineages were present in the Barents Sea and were evenly distributed between the northern waters around Svalbard archipelago and the southern part near Murmansk coast of Kola Peninsula. Both regions, as well as other parts of the O.sarsii range, were characterized by high haplotype diversity with a significant number of private haplotypes, being mostly satellites to the two dominant haplotypes, each belonging to a different mtDNA clade. Demographic analyses indicated that the demographic and spatial expansion of Ophiura sarsii in the Barents Sea most plausibly has started during the Bølling–Allerød interstadial, during the deglaciation of the western margin of the Barents Sea.

Due to the variation of the body coloration and the similarity of the related species, there are still some unresolved problems and debates in the taxonomic study of the genus Sycanus in China. We conducted the inter-specific phylogenetic analyses and the species delimitation for Sycanus in China based on DNA barcoding dataset of the mitochondrial cytochrome c oxidase subunit I gene of 81 samples and partial 28S rDNA sequences of 34 samples. The result showed that all the samples were divided into twelve species by integrating the results of DNA barcoding and morphological comparison. In the present paper, the genus Sycanus in China was reviewed systematically, all species were described or re-described, keyed, illustrated and photographed, among them, Sycanus taiwanensis Zhao & Cai sp. nov., Sycanus flavicorius Li & Cai sp. nov. and Sycanus hainanensis Wang & Cai sp. nov. were described as new to science. The biological informations of Sycanus falleni Stål, 1863 and Sycanus croceus Hsiao, 1979 were briefly recorded.

Spodoptera frugiperda is an invasive pest that has spread in various parts of the world. These pests have the ability to spread and adapt highly to new habitats. Until now, it is not known with certainty the distribution of these invasive pests in Eastern Indonesia, especially Bali and Nusa Tenggara. This study aims to map the spatial distribution and genetic distribution of S. frugiperda which damages maize in the areas of Bali and Nusa Tenggara. This research was conducted using a survey method from May to September 2022 covering the islands of Bali, Lombok, Sumbawa, Sumba, Flores, and Timor. The results showed that S. frugiperda had spread evenly in Bali and Nusa Tenggara. The results of PCR amplification in the COI gene from 9 sample isolates from all research locations showed the similarity of DNA bands leading to the Spodoptera frugiperda species with a banding pattern length of 683 – 697. These results indicated that the distribution of genetic variants of corn caterpillars in Bali, NTB, and NTT was confirmed as S. frugiperda species. However, the isolated gene S. frugiferda, which was shown by the alignment results of the sequences from Lombok, was confirmed as a different strain from strains from Bali, Sumba, Sumbawa, Flores, and Timor. This incident can be seen from the difference in the protein base composition of S. frugiperda from Bali, Sumba, Sumbawa, Timor, and Flores. The results of phylogenetic analysis in this study confirmed 3 clusters of the genetic closeness of S. frugiperda. Cluster-1 comes from the results of the search for specimens of JB FAW and KB FAW from Bali, SB FAW and SB FAW Sorghum from Sumba, SW FAW from Sumbawa, KP FAW from Timor, and FL FAW from Flores. Cluster-2 is an isolate outside of our species. Cluster-3 comes from the search for LT and LS FAW specimens from Lombok. The genetic distance between cluster-1 and cluster-3 is quite far, which is 0.20 mu.

On-going intensification of rice production systems in Southeast Asia is causing devastating yield losses each year due to rice hoppers. Continuing development of immunity to resistant rice varieties and pesticide application further complicate this problem. Hence, there is a high demand for biological control agents. Egg parasitoid wasps are among the most important natural enemies of rice hoppers such as Nilaparvata lugens and Nephotettix spp. However, our knowledge on their diversity is still very limited due to their small size and the lack of available morphological information. Classifying these parasitoids is the first step to properly understand their role in the rice agroecosystem. We used traditional morphological identification as well as DNA sequencing of COI and 28S genes to investigate the diversity of four important hopper egg parasitoid genera in the Philippines. Parasitoids of the genera Anagrus spp., Oligosita spp., Gonatocerus spp. and Paracentrobia spp. were collected in eight study landscapes located in Luzon. We found discrepancies between the morphological and the molecular analysis. Morphological and molecular results were only valid for Paracentrobia spp. Anagrus spp. and Gonatocerus spp. showed more genetic diversity, than expected after the morphological analysis, indicating cryptic species. The sequences for Oligosita spp. revealed less variation than expected. This is the first study on molecular diversity of rice parasitoids in the Philippines. More research combining morphological, behavioural and genetic methods as well as the establishment of a comprehensive DNA database is urgently needed to assess the performance and suitability of these organisms as biocontrol agents.

DNA barcoding has emerged as a powerful tool in the field of taxonomy, transforming the way we identify and classify species. This review article aims to provide an up-to-date overview of the advancements in DNA barcoding techniques, their applications, and their impact on taxonomy and biodiversity studies. We will explore the evolution of DNA barcoding, the development of standardized barcode regions, and the challenges and limitations associated with this methodology. Additionally, we will delve into the diverse range of taxonomic groups for which DNA barcoding has been applied, highlighting key case studies and their contributions to our understanding of biodiversity. Furthermore, we will discuss the integration of DNA barcoding with other molecular and bioinformatic approaches, such as metabarcoding and machine learning, to enhance species identification and biodiversity assessment. This review aims to provide a comprehensive perspective on how DNA barcoding is reshaping the field of taxonomy and its implications for conservation, ecology, and evolutionary biology.

Gobies and their ectoparasitic monogenean flatworms are promising models for species diversification because of their species richness. Recent decades have seen the discovery of several new species of Gyrodactylus (Monogenea: Gyrodactylidae) on European gobies, mostly in the sand goby lineage and especially in the Eastern Mediterranean. However, the monogenean fauna of other gobies is much less understood. Therefore, we scrutinized five gobiid species (34 specimens, vouchered, with some representatives sequenced), sampled in Greece, for monogenean ectoparasites. Only specimens of the giant goby Gobius cobitis were infected; they harbored Xenoligophoroides cobitis (Monogenea: Dactylogyridae) on their gills. Here we provide the first record from Greece, and the first ITS rDNA and COI sequences of the representative of this monotypic genus. Also 28S rDNA was sequenced and compared with published data from across its known distribution, suggesting clinal variation. Lack of sequence data of closely related dactylogyrid monogeneans prevents either proposing a sister-group for Xenoligophoroides, or inferring a scenario explaining the presence of a single known member of this genus on gobies. Possible hypotheses include either the ancestral long-term presence on gobiids but “missing the boat” of the diversification events in the “Gobius-lineage”, or a recent host switch from a non-gobiid host.

In our work, using the analysis of DNA barcodes and morphology (wing color, male genitalia, sphragis shape in females), we show that the group of species close to P. mnemosyne is represented by two phylogenetic lineages, the western and eastern ones. The eastern lineage includes P. stubbendorfii, P. glacialis and P. hoenei. The western lineage includes three morphologically similar species, P. mnemosyne (Western Eurasia), P. turatii (southwestern Europe) and P. nubilosus stat. nov. (Turkmenistan and NE Iran), as well as P. ariadne (Altai). The latter species differs from the rest of the group in the presence of red spots on the wings. Parnassius mnemosyne s.s. is represented by four differentiated mitochondrial clusters that show clear association with specific geographic regions. We propose to interpret them as subspecies: P. mnemosyne mnemosyne (Central and Eastern Europe, N Caucasus, N Turkey), P. mnemosyne adolphi (the Middle East), P. mnemosyne orientalis (Tian Shan) and P. mnemosyne gigantea (Gissar-Alai in Central Asia). We demonstrate that in P. ariadne the red spots on the wing evolved as a reversion to the ancestral wing pattern. This reversion is observed in Altai, where the distribution areas of the western lineage, represented by P. ariadne, and the eastern lineage, represented by P. stubbendorfii, overlap. These two species hybridize in Altai, and we hypothesize that the color change in P. ariadne is the result of reinforcement of prezygotic isolation in the contact zone. Lectotype of Parnassius mnemosyne var. nubilosus Christoph, 1873 is designated.

DNA barcoding has emerged as a transformative tool in the realm of biodiversity assessment, facilitating rapid and precise species identification. This review article delves into the evolving landscape of DNA barcoding, with a particular focus on the emergence of mini-barcodes and alternative genes as innovative approaches. Traditional DNA barcoding, anchored in the COI gene, has significantly advanced our understanding of species diversity; however, inherent challenges persist, especially when dealing with degraded samples or taxonomic groups where COI falls short. The introduction of mini-barcodes, characterized by their reduced sequence length, has revolutionized DNA barcoding by expanding its applicability. We explore the utility of mini-barcodes in various scenarios, ranging from DNA extraction from ancient or fragmented specimens to in-field identification using portable sequencing technologies. Case studies and practical insights into their effectiveness are presented, emphasizing their role in overcoming traditional limitations. Alternative genes, a complementary facet of DNA barcoding, offer a novel avenue for enhancing taxonomic resolution and accuracy. This review showcases instances where researchers have harnessed alternative genes to overcome COI's constraints, particularly in diverse taxonomic groups, such as fungi, plants, and microorganisms. The selection of these genes is discussed, along with their potential to uncover hidden biodiversity and improve species delimitation. Examining the broader implications of mini-barcodes and alternative genes, we unveil their pivotal roles in advancing conservation efforts, forensic science, and citizen science initiatives. The adaptable nature of these approaches empowers a diverse range of stakeholders, from scientists conducting critical research to concerned citizens contributing to our collective understanding of the natural world. Looking ahead, we envision an exciting future for DNA barcoding, as these innovations continue to evolve. Integration with cutting-edge sequencing technologies, data analysis methods, and the harnessing of big data hold immense potential. Nonetheless, challenges related to standardization, scalability, and data sharing persist.

Mandevilla (Apocynaceae) is a greatly appreciated genus in the world ornamental market. In this study, we attempted to address the poor genetic knowledge and the huge taxonomic gaps existing in this genus by analyzing the germplasm of 55 accessions. After cytometrically determining the triploid genome size (1,512.64 Mb) of a reference sample (variety “Mandevilla 2001”), the plastidial genome (cpDNA, 0,18 Mb) and a draft of the nuclear genome (nuDNA, 207 Mb) were assembled. While cpDNA was effective in reconstructing the phylogenesis of the Apocynaceae family based on a DNA superbarcoding approach, the nuDNA assembly length was found to be only 41% of the haploid genome size (506 Mb, predicted based on the K-mer frequency distribution). Its annotation enabled the prediction of thousands of genes, of which 5,275 resulted in full CDS. Among them, we identified nine genes whose orthologs (in Catharanthus roseus) encode enzymes involved in the biosynthesis of monoterpene indole alkaloids (MIAs), including catharanthine, tabersonine and vincadifformine. The nuclear genome draft was also useful to develop a highly informative (mean PIC=0.62) set of 23 SSR markers that was validated on the Mandevilla collection. These results were integrated with cytometric measurements, nuclear ITS1 haplotyping and chloroplast DNA barcoding analyses to assess the origin, divergence and relationships existing among the 55 accessions object of the study. As expected, based on the scarce information available in the literature, the scenario was extremely intricate. A reasonable hypothesis is that most of the accessions represent interspecific hybrids sharing the same species as maternal parent (i.e., Mandevilla sanderi).

The identification of floral visitation by pollinators provides an opportunity to improve our understanding of the fine-scale ecological interactions between plants and pollinators, contributing to biodiversity conservation and promoting ecosystem health. In this review we outline the various methods which can be used to identify floral visitation, providing a comparison between molecular and non-molecular methods. We review the literature covering the ways in which DNA metabarcoding has been used to answer ecological questions relating to plant use by pollinators and discuss the findings of this research. We present detailed methodological considerations for each step of the metabarcoding workflow, from sampling through to amplification and finally bioinformatic analysis. Detailed guidance is provided to researchers for utilization of these techniques, emphasizing the importance of standardization of methods and improving the reliability of results. Future opportunities and directions of using molecular methods to analyze plant-pollinator interactions are then discussed.