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Helobdella aff. robusta Shankland, Bissen & Weisblat, 1992 is recorded for the first time from Japan. The haplotype of the cytochrome c oxidase subunit I (COI) gene sequence of a specimen from Japan is identical to that of one collected in California, USA. The dorsal pigmentation pattern of the Japanese specimens is different from that of H. robusta inhabiting North America, but nonetheless, most of their morphological characteristics are con-sistent with the diagnostic features of this species. This species is considered to be of New World origin, and the Japanese population was likely established from a recent introduction.

Molecular appraoch for identification of unknown species by using Cytochrome b gene is an effective and reliable as compared with morphological based identification. For DNA barcoding universal molecular genes were used to identify the species. Cytochrome b is a specific gene used for identification purpose. DNA barcoding is a reliable and effective method compared to the different traditional morphological methods of specie identification. So,in the present study which was conducted to identify the species, a total of 50 fish samples were collected from five different sites. DNA was extracted by using the Phenol Chloroform method from muscle tissue. Five sequences were sequenced (one from each site), analyzed, and identified specific species as Pangasius pangasius. Identified sequences were variable in length from 369 bp (Site 1), 364 bp (Site 2), 364 bp (Site 3), 352 bp (Site 4), and 334 bp (Site 5). Identity matches on the NCBI database confirmed the specific specie as P. pangasius. A distancing tree was drawn to show maximum likelihood among the same and different species. Yet, in many cases fishes on diverse development stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative tool for species identification and phylogenetic study. This work intends to provide an updated and extensive overview on the DNA based methods for fish species identification by using Cytochrome b gene as targeted markers for identification purpose. Resumo A abordagem molecular para identificação de espécies desconhecidas usando o gene citocromo b é eficaz e confiável em comparação com a identificação baseada na morfologia. Códigos de barras de DNA de genes moleculares universais foram usados para identificar as espécies. O citocromo b é um gene específico usado para fins de identificação. O código de barras de DNA é um método confiável e eficaz em comparação com os diferentes métodos morfológicos tradicionais de identificação de espécies. Assim, no presente estudo, que foi realizado para identificar as espécies, um total de 50 amostras de peixes foram coletadas em cinco locais diferentes. O DNA foi extraído usando o método Fenol Clorofórmio do tecido muscular. Cinco sequências foram sequenciadas (uma de cada local), analisadas e identificadas espécies específicas, como Pangasius pangasius. As sequências identificadas tinham comprimento variável de 369 bp (Local 1), 364 bp (Local 2), 369 bp (Local 1), 364 bp (Local 3), 352 bp (Local 4) e 334 bp (Local 5). As correspondências de identidade no banco de dados do NCBI confirmaram a espécie específica como P. pangasius. Uma árvore de distanciamento foi desenhada para mostrar a máxima probabilidade entre elas e diferentes espécies. No entanto, em muitos casos, peixes em diversos estágios de desenvolvimento são difíceis de identificar por caracteres morfológicos. Os métodos de identificação baseados em DNA oferecem uma adição analiticamente poderosa ou mesmo uma ferramenta alternativa para identificação de espécies e estudo filogenético. Este trabalho pretende fornecer uma visão geral atualizada e abrangente sobre os métodos baseados em DNA para identificação de espécies de peixes usando o gene citocromo b como marcadores direcionados para fins de identificação.

One new species of the genus Pseudopoda Jäger, 2000, Pseudopoda deformis Gong & Zhong, sp. nov. (♂, ♀), is described and documented with digital images from Shennongjia Forestry District, Hubei Province, China, based on morphology and DNA barcodes. This new species is separated from other Pseudopoda species by the unique type of internal ducts of the female vulva that are curved longitudinally, forming a narrow triangle or trapezoidal shape. In addition, DNA barcodes for this species are provided.

Currently, the genus Arctia Schrank, 1802 includes approximately 16 species in the Palaearctic region, depending on the taxonomic interpretation. Here, populations of the Arctia villica (Linnaeus, 1758) morphospecies complex were studied from Europe to the Middle East (Turkey, northern Iran) by molecular methods. Morphological treatment has traditionally revealed the presence of five nominal taxa: A. villica (Linnaeus, 1758), A. angelica (Boisduval, 1829), A. konewkaii (Freyer, 1831), A. marchandi de Freina, 1983, and A. confluens Romanoff, 1884. The molecular approach tests whether they represent well-delimited species. Subsequently, this study corroborates the suitability of the mitochondrial cytochrome c oxidase subunit 1 (COI) marker sequence for species delimitation. In total, 55 barcodes of the Arctia villica complex were compared, and two molecular species delimitation algorithms were applied to reveal the potential Molecular Operational Taxonomic Units (MOTUs), namely the distance-based Barcode Index Number (BIN) System, and the hierarchical clustering algorithm based on a pairwise genetic distances approach using the Assemble Species by Automatic Partitioning (ASAP). The applied ASAP distance-based species delimitation method for the analysed dataset revealed an interspecific threshold of 2.0–3.5% K2P distance as suitable for species identification purposes of the Iberian A. angelica and the Sicilian A. konewkaii and less than 2% for the three taxa of the A. villica clade: A. villica, A. confluens, and A. marchandi. This study contributes to a better understanding of the taxonomy of the genus Arctia and challenges future revision of this genus in Turkey, the Caucasus, Transcaucasia as well as northern Iran using standard molecular markers.

The family Lecithoceridae is not well represented in the Palaearctic region, with very few taxa in Europe. Here we describe a new genus and species of lecithocerid moth, Xenotorodor stygioxanthus Sterling, Lees & Grundy, gen. nov., sp. nov. The taxon represents a subfamily new to Europe. We consider placement of the genus within Crocanthinae or Torodorinae. We place it in Torodorinae, notwithstanding the reduced gnathos in the male genitalia. DNA barcodes suggest that the taxon belongs within a distal clade of this subfamily. They are over 9.2% pairwise divergent from any hitherto cleanly sequenced Lepidoptera taxon and over 10.1% from nearest taxonomically identified neighbours in Neighbor Joining and ML trees. Characteristics of the DNA barcode and morphology of this new taxon suggest that refinement of synapomorphies for the family and two subfamilies is needed. We have nearly 100 records for this new species since 2020, all from a small area of Southern Spain, close to the Straits of Gibraltar. The life history and early stages of the species are unknown.

The snorkel snail genus Rhiostoma Benson, 1860 is comprised of terrestrial cyclophorid snails with wide-ranging species diversity and radiation in Southeast Asia. The typical characters of the genus are a depressed shell, a detached and descending portion of the last whorl with a distinctive peristomal breathing device attached, and a calcareous cup-shaped operculum. Herein, we have revised the systematics of extant species based on shell morphology combined with COI barcoding. From these thirty recognised species, twelve are described as new to science: R. ? amarapuraensesp. nov., R. anceyisp. nov., R. breviocollarsp. nov., R. ebenozosterasp. nov., R. cheliopegmasp. nov., R. furfurosumsp. nov., R. gnomus, sp. nov., R. lannaensesp. nov., R. laoensesp. nov., R. platymorphasp. nov., R. rhothonotaphrosasp. nov., and R. tigrinasp. nov. All conchological characters are provided via illustrations of type specimens and living snails, and descriptions of the shells and radulae. Phylogenetic analysis based on partial COI gene sequences strongly supports the designated morphospecies and a monophyletic Rhiostoma, confirming that all pterocyclinid snails with a calcareous, cup-shaped operculum belong to the same clade. A high intra-specific divergence was observed in R. jalorensis and R. housei populations from locations in close proximity, suggesting a lower dispersal and higher level of isolation. The low inter-specific divergence found in R. hainesi, R. samuiense, R. asiphon, and R. rhothonotaphrosasp. nov. supports their recent diversification and local adaptation, and is congruent with their marked morphological differences. Finally, nine formerly Rhiostoma-placed species were reclassified into either the genus Cyclotus or the genus Opisthoporus.

1. DNA barcoding aids in rapidly identifying specimens in various contexts by comparing short stretches of DNA to a reference database. Barcoding initiatives generate large reference databases of carefully curated high‐quality sequences to maximize identification success. 2. BarKeeper provides a flexible tool for barcoding initiatives and in the context of phylogenetic studies to foster shared work on large datasets of raw sequence and associated metadata. It is free, open‐source and available as a set of Docker containers for easy setup. After setting it up once, all project members can use it independently of their operating system or location. 3. BarKeeper offers features to collect and manage data and metadata about specimens, taxa and DNA sequences from Sanger sequencing and high‐throughput sequencing (HTS) technologies. It provides excellent flexibility by not being restricted to specific markers or taxon groups. Users can view and edit records and associated metadata while the app assists them by trimming and assembling reads. It automatically checks the quality of generated barcodes with a taxonomy‐based tool and offers a wide range of options for data analysis. Extensive search features allow querying the database for specific groups of records and saving the search results in the user's profile or downloading them in various file formats. 4. BarKeeper combines multiple tools to aid barcoding projects in every step, from a reference taxa list to finished barcode sequences, thereby minimizing the number of laborious, potentially error‐prone manual steps and enabling efficient collaborative workflows.

We recently collected the samples of Henricia from adjacent waters of Dokdo Island, Korea, using trimix SCUBA diving. Based on a combined result of morphological and molecular analysis, we identified our specimen as Henricia djakonovi Chichvarkhin, 2017, which is newly recorded in Korea. Morphologically, H. djakonovi has crescent abactinal plates bearing numerous pillar-shaped abactinal spines with a droplet-like apical tip. Moreover, molecular analysis based on the mitochondrial COI gene occurred that clearly distinguished H. djakonovi from other species of Henricia in the pairwise genetic distance and maximum likelihood analysis. Accordingly, 15 species of Henricia are recorded in Korean fauna, including H. djakonovi.

広島大学東広島キャンパスには多くの樹木が植栽されている。2020 年度から，学内の植物相を明らかにする一環で植栽樹木の調査を実施している。調査の結果，169 種6,535 本の植栽樹木が確認された。また，植栽樹木の中に絶滅危惧種が確認された。環境省のレッドリストで絶滅危惧II 類に選定されているハナノキAcer pycnanthum K.Koch（ムクロジ科）が確認された。樹木などの場合，生育にはある程度広い空間が必要であるが，キャンパスのような場所は域外保全の場としても有用であると考えられる。また，調査の結果確認された代表的な樹種による二酸化炭素固定量を計算したところ，年間炭素固定量は31.65 t であった。二酸化炭素削減の観点からもキャンパスの樹木の重要性が示唆された。, Many tree species are planted on the Higashi-hiroshima Campus of Hiroshima University (Hiroshima Prefecture, SW Japan). Since 2020, multiple surveys have been conducted to clarify the flora here. We confirmed 6,535 trees belonging to 169 species. The annual carbon dioxide fixation rate of the representative trees identified in the study was 31.65 tons. We also identified Acer pycnanthum K. Koch (Sapindaceae), which is listed as a vulnerable species in Japan’s Red List. As tree species require a certain amount of space for growth, we consider sites such as school campuses useful for ex situ conservation and campus trees important for social amenities and carbon dioxide reduction.

To improve breeding techniques for aquarium freshwater pufferfishes, hormone injection (hCG, 10 IU per g)-induced maturity and insemination of Auriglobus modestus from Thailand were undertaken, and morphological development of eggs and early stage larvae observed. Artificial insemination was performed two to five days after hormone injection. Although no fertilized eggs resulted from artificial insemination utilizing a wet (freshwater) method, an isotonic (sodium lactate ringer solution) method produced 458 fertilized eggs out of 787 eggs (fertilization rate 61.7%) spawned by one female, although only 8 larvae (1.7%) eventually hatched. Eggs were oval [long axis 2.05 ± 0.06 mm, short axis 1.68 ± 0.06 mm (n = 5)], translucent, demersal, and adhesive, and contained a number of small yellow oil globules. Oval eggs are unusual among pufferfishes, although common in the genus Chonerinos, an indication of the genetic closeness of the latter and Auriglobus, but also suggesting ecological similarity. The eggs hatched after 2 days, larvae one day after hatching [4.54 ± 0.34 mm NL (n = 9)] having a large yolk sac. However, the mouth remained unopened, the optic vesicle uncolored, and the pectoral fin membrane undeveloped, except for small knob-like rudiments. Seven days after hatching, larvae [5.22 ± 0.10 mm NL (n = 3)] had lost the yolk sac and acquired rudimentary soft rays in the pectoral, dorsal and anal fins. All larvae died after eight days. DNA barcoding comparisons (COI gene, 652 bp) of several pufferfish genera indicated a close genetic relationship of Auriglobus and Chonerhinos, being nested in the same clade.

Salmon is a popular fish with high nutritional and economic value due to its delicious and tender pink flesh rich in omega-3. However, there is a problem with mislabeling other types of fish as salmon in the market, which is misleading for consumers. A study used molecular methods to identify fish species sold as salmon in commercial centers and found that out of the 35 samples analyzed, 33 were indeed salmon, but two were trout. The study highlights the need for strict enforcement of food trade and labeling regulations.

Here, we describe and illustrate Encelia balandrasp. nov., a new species of Compositae from the Baja California Peninsula. It is rare and known only from the rocky hills around Puerto Balandra and Pichilingüe, inside the bay of La Paz, in the State of Baja California Sur, Mexico. We determine that this new species has affinities with Encelia, based on its suffruticose woody habit, neuter ray florets and compressed disc cypselae with a cleft apex. The taxonomic placement within Encelia is supported by nuclear ribosomal sequence data from two regions, ITS and ETS. We also present detailed photographs, a conservation assessment and a dichotomous key to the Encelia of the southern Baja California Peninsula. Finally, we discuss the uniqueness of Encelia balandra amongst peninsular Encelia and its potential significance for understanding the enigmatic biogeography of this ecologically important genus.

A new speciesLeioproctus zephyr(Hymenoptera: Colletidae) is described from both sexes.Leioproctus zephyrsp. nov.is remarkable in featuring a large longitudinal ridge on the clypeus. This diagnostic morphological feature present in both sexes, along with various other distinctive characters including the male genitalia, female hind-tibial spur, and glossa morphology, clearly distinguish this species from all otherLeioproctus. Along with these unique traits,L. zephyrcannot be classified into any of the existing subgenera ofLeioproctus, sharing some, but not all, of the characters of the subgeneraCeratocolletes,Charicolletes,ProtomorphaandOdontocolletes. DNA barcoding with the CO1 gene confirmed the sexes belonged to the same species and it did not match any previously barcoded species. This species is restricted to native vegetation remnants in the southwest Western Australian biodiversity hotspot, and is highly specialised, foraging only on a few species in the genusJacksonia(Fabaceae). The unusual clypeus may be an adaptation for foraging on the keeled papilionaceous flowers. The limited number of sites this species has been collected from and its oligolectic diet suggestL. zephyrshould be considered to be a species of conservation concern. Further taxonomic research is required to determine the phylogenetic position of this unusualLeioproctus.

Two new species of tetragnathid spiders from Guizhou and Sichuang provinces of China are described: Diphya guiyang J. Zhang & H. Yu, sp. nov. (♂♀) and Diphya weimiani J. Zhang & H. Yu, sp. nov. (♀). Detailed descriptions, diagnoses, and photographs are provided for these two species, as well as a key and a distribution map for Chinese Diphya species. DNA barcodes (a partial fragment of the mitochondrial cytochrome oxidase subunit I gene, COI) of both new species were obtained for species delimitation, matching of different sexes, and future use in molecular studies.

The DNA of Trigonopterus specimens from the Sundaland region stored between ten and 32 years in museums could be used for next-generation sequencing. The availability of their cox1 sequence allowed the description of the following nine new species: Trigonopterus grimmisp. nov., T. johorensissp. nov., T. lambirensissp. nov., T. linauensissp. nov., T. microreticulatus Riedel, Trnka & Wahab sp. nov., T. mulensissp. nov., T. sarawakensissp. nov., T. siamensissp. nov., and T. singaporensissp. nov. The alternative original spelling of the name T. tounensis Narakusumo & Riedel is chosen to prevail over T. tounaensis Narakusumo & Riedel. The new species represent the first country records of Trigonopterus for Brunei, Singapore, and Thailand. Thus, the genus´ known area of distribution in the Sundaland region is significantly extended. A key and a catalogue are provided to the Trigonopterus species from Borneo, W-Malaysia, Singapore, and Thailand.

The genus Staurolemma Körb., represented by the species S. omphalarioides (Anzi) P. M. Jørg. et Henssen, identified both by morphology and using DNA barcoding (ITS1-5.8S-ITS2 gene), is reported for the first time for Russia, based on materials collected in the North-Western Caucasus in the Utrish Reserve on the Abrau Peninsula. The genus Staurolemma is widely distributed in the tropics and subtropics, and only one species S.omphalarioides, has a Mediterranean-Atlantic area. As in the Mediterranean basin, in the Abrau Peninsula, the species is confined to warm and humid habitats on the Black Sea coast, where it grows in sub-Mediterranean plant communities, in oak-juniper and juniper-pistachio forests, on the trunks and branches of deciduous trees, mainly on Pistacia mutica Fisch. et C. A. Mey. and Quercus pubescens Willd. Description and photo of species are given with a discussion on their taxonomy and distribution. For some studied specimens, new rITS sequences were generated and their GenBank accession numbers are provided., Род Staurolemma Körb., представленный видом Staurolemma omphalarioides (Anzi) P. M. Jørg. et Henssen, впервые приводится для России по сборам из Северо-Западного Кавказа с территории заповедника «Утриш» на п-ове Абрау. Виды рода Staurolemma широко распространены в тропических и субтропических областях Земли, и только один вид S. omphalarioides имеет средиземноморско-атлантический ареал. Как и в средиземноморском бассейне, на п-ове Абрау вид приурочен к теплым и влажным местообитаниям на Черноморском побережье, где произрастает в субсредиземноморских растительных сообществах, в дубово-можжевеловых и можжевелово-фисташковых лесах, на стволах и ветвях лиственных деревьев, главным образом, на Pistacia mutica Fisch. et C. A. Mey. и Quercus pubescens Willd. В статье приводятся описание и фото вида, а также информация о его систематическом положении и географическом распространении. Для части изученных образцов секвенированы нуклеотидные последовательности ITS-участка рДНК (их номера в базе данных GenBank указаны в тексте).

Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market. Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation. Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2°C on average. Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.

Landscape changes, over time, lead to changes of floral resources available to pollinators, which in turn may result in the disappearance of ecologically specialized species. Here, we use pollen metabarcoding to infer historic and recent interactions between plants and bumblebees (Bombus). Bumblebees from Cuxhaven (Germany) were sampled from historical museum collections (1968/69) and in the field (2019). Pollen attached to their bodies was barcoded using multiple plant markers (ITS1, ITS2 and trnL-P6 loop). Our results show shifts in foraging habits between the historic and recent sampling periods, mostly determined by fewer Fabaceae interactions in 2019. The successful implementation of scalable molecular techniques for the analysis of historical pollen samples underscores the value of museum collections as a resource for biodiversity research. This study provides proof of concept of a comparative analysis of recent and historical pollination data. However, to ensure the robustness of our results, it is crucial to consider the broader methodology used. Our study found variation in the efficacy of the three plant barcoding markers. The ITS1 marker exhibited the highest species-level identification success, while the trnL-P6 loop demonstrated utility in amplifying degraded DNA across diverse plant families.

The ability to correctly identify specimens at the species level is crucial for assessing and conserving biodiversity. Despite this, species-specific data are lacking for many of South Africa’s catsharks due to a high level of morphological stasis. As comprehensive and curated DNA reference libraries are required for the reliable identification of specimens from morphologically similar species, this study reviewed and contributed to the availability of cytochrome c oxidase subunit I (COI) and nicotinamide adenine dehydrogenase subunit 2 (NADH2) sequences for South Africa’s catsharks. A molecular taxonomic approach, implementing species delimitation and specimen assignment methods, was used to assess and highlight any taxonomic uncertainties and/or errors in public databases. The investigated species were summarised into 47 molecular operational taxonomic units (MOTUs), with some conflicting specimen assignments. Two Apristurus specimens sampled in this study remained unidentified, revealing the presence of previously undocumented genetic diversity. In contrast, haplotype sharing within Haploblepharus—attributed to nucleotide ambiguities—resulted in the delimitation of three congeners into a single MOTU. This study reveals that molecular taxonomy has the potential to flag undocumented species and/or misidentified specimens, and further highlights the need to implement integrated taxonomic assessments on catsharks that represent an irreplaceable component of biodiversity in the region.

This study aimed to explore the applicability of DNA barcoding for assessing the authenticity of caviar on the Chinese market. A set of universal COI primers and two sets of designed primers based on COI and D-loop genes were used to identify maternal species of samples from 21 batches of caviar. The results showed that the PCR products from three sets of primers had more than 98% similarity to the sequences in database. The COI gene could not distinguish sturgeons with closed genetic relationships, while D-loop gene could effectively improve the accuracy of DNA barcoding and was more suitable to the identification of interspecific sturgeon than the COI gene. The neighbor-joining dendrogram further confirmed the applicability and accuracy of COI and D-loop genes in identifying maternal relatives of caviar (Acipenser baerii/Acipenser gueldenstaedtii/Acipenser schrenckii/Huso dauricus/Huso huso). Despite the limitations of mitochondrial DNA in identifying hybrid sturgeon species, the presence of counterfeit caviar of non-sturgeon ingredients could be excluded. All the caviar samples were identified successfully as sturgeon species, but the mislabeling rate of species was 33.4%, indicating that there were illegal phenomena such as disorderly labeling, mislabeling, and adulteration on the market.

The use of DNA has helped to improve and speed up species identification and delimitation. However, it also provides new challenges to taxonomists. Incongruence of outcome from various markers and delimitation methods, bias from sampling and skewed species distribution, implemented models, and the choice of methods/ priors may mislead results and also may, in conclusion, increase elements of subjectivity in species taxonomy. Lack of direct diagnostic outcome from most contemporary molecular delimitation approaches and need of reference to existing and best sampled trait reference systems, reveals the need of refining criteria of species diagnosis and diagnosability in the current framework of nomenclature codes and good practices to avoid nomenclatorial instability, parallel taxonomies, and consequently more and new taxonomic impediment.

Subterranean arthropod communities are important components of North American ecosystems, contributing numerous ecosystem services and essential food-web functions. Despite this, fundamental information about species diversity in these communities remains unknown, and their taxonomic composition and ecological diversity have scarcely been assessed. Subterranean pitfall traps are a commonly used method for sampling endogean and hypogean soil habitats in Europe but have never been widely implemented in North America. Here, I employed this method to sample subterranean arthropod communities in the Ridge and Valley and Blue Ridge ecoregions of Virginia, USA in the winter and spring. In total, 2,260 arthropod specimens were collected constituting 319 distinct species. I extracted and purified DNA and amplified the mitochondrial gene: cytochrome C oxidase subunit I (COI) from each recovered morphospecies and derived a unique COI barcode for each species sequenced. Objective sequence clustering was used to establish molecular operational taxonomic units (mOTUs) for downstream diversity analyses and establishment of dynamic identification resources. Total species richness and average species richness per site were assessed and compared for both regions and seasons. The Shannon-Wiener diversity index, Hutcheson's t-test, and effective numbers of species (ENS) were employed to compare regional subterranean arthropod diversity. The richness, Shannon-Wiener, and ENS comparisons indicated that both ecoregions encompass highly diverse subterranean arthropod communities with those of the Ridge and Valley being significantly more diverse than those of the Blue Ridge. Master of Science in Life Sciences Arthropods are invertebrate animals with hard exoskeletons, segmented bodies, and jointed paired appendages, and include insects, arachnids, crustaceans, and myriapods. Arthropods make up the majority of animal species on the planet and are important parts of ecosystems, making it important for researchers to study them. While we know a fair amount about North American arthropods that live above-ground, relatively little is known about those that live deep within the soil and underlying rock substrate. Subterranean pitfall traps are a common method used for collecting arthropods from subterranean habitats in various regions of the world but have rarely been used in the USA. As a result, much of the life beneath our feet may be unknown. I used these traps to collect subterranean arthropods in the Ridge and Valley and Blue Ridge regions of the Appalachian Mountains. A total of 2,260 individual arthropods were collected, belonging to 319 different species. I used DNA sequencing to establish a unique fingerprint-like "barcode" for each species. These barcodes serve as helpful identification resources, and will help name new species in the future. They also allowed me to measure the number of species (diversity) of subterranean arthropods collected from each region, and compare the two to determine which region is more diverse. I used common statistical metrics of diversity including species richness (number of species) and the Shannon-Wiener diversity index to compare regional subterranean arthropod diversity. My results show that both the Ridge and Valley and Blue Ridge regions of Virginia, USA are home to highly diverse subterranean arthropod communities and those of the Ridge and Valley are significantly more diverse than those of the Blue Ridge.

Pangasiids are an economically significant group of catfish, and many pangasiids are threatened in the wild from anthropogenic pressures, including increases in fishing pressure, habitat degradation, and improperly managed aquaculture practices. This study demonstrates the usage of DNA barcoding of the Cytochrome Oxidase subunit I (COI) gene as an identification tool in detecting potentially threatening invasive pangasiid species by establishing the diversity and phylogenetic relationship of Pangasiidae catfishes in Peninsular Malaysia. A neighbour-joining (NJ) dendrogram (Kimura-2-parameter model) generated five clades to represent distinct genera. Pangasius was further subdivided into two clades (Clade A: Pangasius bocourti-P. djambal and Clade B: P. nasutus-P. conchophilus). Given the marginal genetic divergence, indigenous and non-native species should be treated cautiously in allopatrically distributed species. The analysis used Automatic Barcode Gap Discovery (ABGD) and revealed barcode gaps between the intraspecific and interspecific distances. The sequences were partitioned into five groupings, corresponding with the species delineation based on the distribution of pairwise differences, which could not be differentiated using the NJ dendrogram. ABGD allows the recognition of one or two additional species using the recursive approach, but other taxonomic methods should be considered for a solid conclusion. DNA barcoding demonstrates the identification of closely related species, thus justifying its application towards the conservation of these fish.

The marine gastropod mollusc Nassarius sinarum has attracted attention due to its status as a potential invasive species and the ecological impact it may have on local environments and the fishing industry. It was observed exclusively within China initially, but its distribution now seems to have expanded into Japan and Korea. Accurate identification of N. sinarum, particularly in its juvenile stage, is vital for understanding its ecological influences and distribution patterns.This study represents the first comprehensive analysis of N. sinarum samples from Korea. It includes morphological examination, scanning electron microscopy images and molecular sequencing. Two live specimens were collected from the Yeongsan River estuary in Korea and their morphological features were analysed and compared to those of samples from China and Japan. The samples' species were confirmed by molecular identification, based on cytochrome c oxidase subunit I (COI) and histone H3 (H3) genetic markers.It was observed that juvenile N. sinarum shells lack key species-characteristic morphological traits, such as a thick outer lip and diminishing axial ribs. However, COI marker-based molecular identification affirmed that these Korean specimens were N. sinarum. The H3 region was registered with the National Center for Biotechnology Information (NCBI) for the first time. Phylogenetic analysis of the H3 region did not resolve species distinctions within the Nassarius, suggesting that the H3 marker is not suitable for species identification within this genus. In this context, multiple genetic markers, when used appropriately, can also be applied to genus-level searches, enhancing species identification accuracy and reducing misidentification.The sequences provided in this study can serve as a valuable reference for future DNA barcoding research. Additional samples and surveys should be conducted through collaborative efforts amongst national and institutional organisations to further clarify the ecological status of N. sinarum and to investigate its distribution and potential impact around East Asia. Finally, a new Korean name, (No-lan-jul-job-ssal-mu-nui-go-dung; 노란줄좁쌀무늬고둥) has been proposed for N. sinarum.

Western Indian Ocean countries have banned elasmobranch finning and enacted legislation to protect endangered elasmobranchs, however finned and morphologically deformed specimens are still landed and traded on Tanzanian fish markets. Such specimens are difficult to identify morphologically, and it is possible that protected elasmobranchs are among these. This study used DNA barcoding to uncover protected elasmobranchs in 102 specimens traded on Tanzanian fish markets. The sampled specimens revealed 23 elasmobranch species, 12 of which (52.1 %) were classified as endangered (EN) or critically endangered (CR) on the IUCN Red List. Three of the identified species (great hammerhead Sphyrna mokarran, oceanic whitetip shark Carcharhinus longimanus, and pelagic thresher Alopias pelagicus) are protected by Tanzanian laws. Hence, it is advised that steps be taken to strengthen law enforcement at landing sites and fish markets in the country. Furthermore, the Third Schedule of Fisheries (Amendment) Regulations of 2009 should be updated to include 11 EN and CR elasmobranchs that are not on the list. Additionally, national and regional elasmobranch conservation plans should be developed to prevent the exploitation of endangered elasmobranchs.

Telling species apart plays a key role in understanding global biodiversity, monitoring change, and managing biodiversity. However, species discrimination is often difficult due to the sheer volume of species on earth and the complexity of the nature of species. This led to the development of DNA barcoding which uses standardised regions of DNA for species identification. The standard plant DNA barcodes are based on small regions from the plastid genome and ribosomal DNA. The approach works well in some plant groups, but does not provide unique species-level resolution in many others. In this thesis, I explore the potential for using data from the nuclear genome for improving and enhancing species discrimination in plants. Access to the nuclear genome via high-throughput sequencing now enables the generation of large amounts of sequence data from multiple unlinked loci. Such data offer the opportunity for designing the next generation of plant barcoding approaches based on a detailed understanding of genomic differences between species. Various studies have shown the ability of multiple unlinked nuclear markers to provide high discriminatory power in many plant groups, separating species, and infra-specific taxa. But there has not yet been an overview and synthesis of exactly how powerful these approaches can be, and how best to guide future efforts in building plant identification tools. In this thesis, I provide a first overview of how nuclear genomes perform in telling species apart. Overall I tackled the following questions 1) what is the proportion of species that are distinguishable with nuclear markers? 2) what is the nature of the inter-specific differences and what are the attributes of loci that are the most informative in telling species apart? And 3) how many markers are needed and what markers are needed to maximise the species identification success? To answer those questions, I first outlined the conceptual issues to address in assembling and analysing appropriate multi-locus nuclear sequence datasets. I then developed a new pipeline called NucBarcoder which supports workflows and analysis using multi-locus nuclear sequence data for species discrimination. I then tested this workflow on a case study, consisting of a dataset of sequence data from 810 nuclear genes from 453 individuals from 133 Inga species including 69 species which were represented by multiple-sampled individuals. Of the 69 species with multiple individuals sampled, 45 resolved as monophyletic (65%). The density of species- specific SNPs for each Inga species ranged from 0 to 1503 per megabase. Compared to the full dataset of 810 genes and 205,871 SNPs, subsampling analysis revealed that a random selection of 70 genes or 2500 SNPs, or a combination of 9 ‘best performing’ genes could achieve levels of species discrimination success similar to the full dataset. I found a positive correlation (r = 0.42) between the number of species distinguished and the nucleotide diversity of the genes used for species discrimination. To search for broader generalisations, I then compiled data from 149 different genera to assess the proportion of plant species that resolve as monophyletic. I then selected 29 genera with suitable available data for further study and calculated the abundance and density of species-specific SNPs (SSSNPs), and the proportion of species that can be distinguished by different subsets of the data and also by targeting the best- performing gene regions. Finally, I evaluated the characteristics of the best-performing gene regions in terms of levels of nucleotide diversity and density of SSSNPs. In the II analysis of 149 genera, overall, of the 1,701 multiple-sampled species evaluated 1,206 resolved as monophyletic (71%). At the level of individual genera, 37 of the 149 genera (25.8%) had 100% of species resolved as monophyletic, and 75 (50.3%) genera had at least 75% of the species resolving as monophyletic. Among the 29 genera examined in more detail, the density of SSSNPs of all species ranged from 0 to 27,262 per Mb, with a median density of 323 SSSNPs per Mb (a median density of one SSSNP every 3,098 bp). Of the total of 460 species from 29 genera assessed, 411 species (89.3%) had at least one SSSNP. Resampling of these datasets showed that with around 3,000 SNPs, almost all genera have asymptoted in their levels of species discrimination, with 21/23 genera (91%) having >85% of their distinguishable species (e.g. those told apart in the full data set) distinguished with 3,000 randomly selected SNPs. Furthermore, in a detailed investigation of six genera, there are clearly some loci that are much better than others in telling species apart. Between one and nine pre-selected genes were able to recover equivalent levels of species discrimination compared to several hundred genes from the full datasets. A closer investigation of the attributes of the best-performing genes showed some positive correlations between the number of species resolved as monophyletic and the nucleotide diversity of a given gene, although this relationship was not clear cut, and the genes that give the highest species resolution are not always the most variable genes. These findings provide some key general information on the proportions of plant species that are resolvable using multi-locus nuclear sequence data from plants and the nature of the sequence variation between plant species. In the final chapter of the thesis, I summarise these findings and identify a set of priority research and infrastructure needs to take forward the development and use of multi-locus nuclear DNA barcoding of plants.

International audience; Aim: The mosaic of savannas that persists in the forest-dominant Congo Basin is thought to be palaeoclimatic relics, but past biogeographical processes that have formed and maintained these systems are poorly understood. Here, we explored the post-Pleistocene biogeography of Gabon's savannas using termites as biological indicators to understand historical and mechanistic factors influencing present-day termite communities in the country's extant savannas. Location Gabon, Central Africa. Taxon Blattodea: Termitoidae. Methods Using standardised transect methods, we sampled termite communities in four disjunct modern savanna areas of Gabon: the centre (Lopé), the southeast (Batéké) and the south (Mayombe North and South). Termites at Lopé were collected in three habitats (annually burned savannas, savannas with a depressed fire regime and forest). We used DNA barcoding of the COII region to identify termite species and compared abundance, species richness and community composition across areas and habitats. Results Community composition differed greatly between Lopé and both Batéké and Mayombe savannas with Lopé being exceptionally depauperate and lacking characteristic savanna species. Within Lopé, termite abundance and diversity was highest in forests and lowest in annually burned savannas, with a gradual change in species composition across the forest–savanna gradient associated with fire history. Main Conclusions The absence of savanna typical species in Lopé savannas challenges current assumptions that these savannas were linked to the south/southeastern savannas during the Pleistocene and suggests a different evolutionary history. Lopé savannas may instead have opened as an isolated grassland and never have been contiguous with neighbouring savannas, or were isolated soon after forest expansion began and have now lost savanna-typical species. Furthermore, the patterns of termite community composition in fire suppressed savannas support a hypothesis of rapid change driven by fire frequency where either fire suppression or infrequent burning over 23 years has meant savannas have become ecologically much more forest-like.

Abstract Piophilidae Macquart, 1835, is a small family of flies widely distributed in all parts of the world but more represented in the cooler temperate regions of the Northern Hemisphere. At present, there is a lack of information on the distribution of this family in the Caucasus region. In this study, the family Piophilidae, with a record of Prochyliza nigrimana (Meigen, 1826), is reported for the first time from the South Caucasus ecoregion.

The genus Nannarrup Foddai, Bonato, Pereira & Minelli, 2003 is a monotypic genus established on the basis of the possibly introduced species N. hoffmani Foddai, Bonato, Pereira & Minelli, 2003, from New York, USA. In the present study, in a field survey conducted throughout Japan, Nannarrup-like specimens were collected from Honshu, Shikoku, and Kyushu. These specimens clearly showed the diagnostic characteristics of the genus but were morphologically distinct from N. hoffmani. Furthermore, morphological analysis and DNA barcoding revealed that these specimens could be assigned to two distinct undescribed species. On the basis of these results, N. innuptus Tsukamoto, sp. nov. and N. oyamensis Tsukamoto, sp. nov. are described. The three Nannarrup species can be distinguished from each other on the basis of the following combination of characteristics: presence or absence of a pair of smooth or weakly areolate areas along the posterior part of the paraclypeal sutures; the width-to-length ratio of the denticle on the trochanteroprefemur; the pigmentation of the denticle on the tarsungulum. Moreover, the field survey resulted in the collection of exclusively female specimens of N. innuptus Tsukamoto, sp. nov., which shows the possibility of parthenogenesis of this species.

The subgenus Brevantennia Sieder, 1953 of the genus Dahlica Enderlein, 1912 (Psychidae: Dahlicini) is represented by a group of 10 bagworm moth species, distributed in south-west and south-east Europe northwards to the Alps and Carpathians. This study is a revision of the subgenus Brevantennia species occurring in Slovenia based on our own comprehensive field studies, DNA barcoding and morphological analysis. Dahlica (B.) santicensis (Sieder, 1957) is established as bona spec. and D. (B.) gorskikotarica Weidlich, 2015, syn. nov. is synonymised with D. (B.) santicensis. Both D. (B.) adriatica (Rebel, 1919) and D. (B.) triglavensis (Rebel, 1919) are confirmed to occur in Slovenia. D. (B.) styriaca (Meier, 1957) is excluded from the checklist of Slovenian Lepidoptera fauna.

Cephalopods form important components of marine trophic systems worldwide, including those in the South Pacific. Moroteuthopsis ingens (Onychoteuthidae) is one of the most abundant deep–sea squids in the Aotearoa New Zealand region and is preyed upon by predators such as the sperm whale and orange roughy. However, despite the many publications on M. ingens’ diet, knowledge on its feeding ecology remains limited. In this study, we analysed the prey of M. ingens from the Chatham Rise (an ecologically and economically important region east of Te Waipounamu/New Zealand’s South Island) using DNA barcoding and otolith identification. Lanternfishes (Myctophidae) were the most frequently encountered prey item, but other teleost species, cephalopods and crustacean fragments were also found, indicating opportunistic and likely cannibalistic feeding behaviour. Five fish species (including a possibly undescribed species of Gymnoscopelus) and Bathyteuthis abyssicola are reported as prey items of M. ingens for the first time. Moroteuthopsis ingens appears to be a component of several dynamic food chains and likely plays an important role in the transfer of carbon throughout marine environments in the Southern Hemisphere.

Baiyuerius gen. nov., a new genus of the subfamily Coelotinae F. O. Pickard-Cambridge, 1893 is described, including five new species: B. daxi sp. nov. (♀), B. pindong sp. nov. (♂), B. tamdao sp. nov. (♀), B. zhuping sp. nov. (♂) and B. zuojiang sp. nov. (♂♀), from southern China and northern Vietnam. Our molecular phylogenetic analyses support Baiyuerius gen. nov. as monophyletic and as a sister group of the newly established genus Yunguirius Li, Zhao & Li, 2023.

The South Caucasus Region and Georgia, in particular, is a biodiversity hotspot and characterised by high diversity of landscapes and ecosystems, as well as high levels of endemism. At the same time, diversity of freshwater organisms in the region remains poorly studied, including fishes. The freshwater fish fauna of the South Caucasus Region consists of 119 fish species, of which 13 species belong to the order Gobiiformes. It should be noted that gobies are amongst the poorly studied taxa in Georgia and probably unknown/undescribed species still living in the Georgian freshwater ecosystems which requires further research.Ponticola alasanicus, a new species is described from the Alazani River, western Caspian Sea Basin, Georgia. It is distinguished from its congeners in the Caspian and Black Sea Basins by having the following features: dorsal fin with VI-VII spines and 15½-16½ branched rays, anal fin with 10½-12½ branched rays; lateral line with 48-55 scales; laterally compressed body with dark brown and black blotches - scales ctenoid; first and second dorsal fins almost touching with dorsal fins bases; head large, depressed, wider than deep, its length approaches almost 3.4th of standard length; nape scaled completely; cycloid scales cover upper part of opercle, cheeks noticeably swollen; snout longer than eye, eye diameter 4.5 times its head length; lower jaw slightly protruding; upper lip is uniform; pelvic disc short, elongated and flat, not reaching the anus; the pectoral fins extends vertically through first branched dorsal fin; caudal fin rounded. Ponticola alasanicus sp. n. belongs to P. syrman group and it is separated by a minimum Kimura 2-parameter distance of 3.5, 3.6 and 4.8% from P. syrman, P. iranicus and P. patimari, respectively.

DNA barcoding based on the cytochrome oxidase I gene is increasingly used in black flies (Diptera: Simuliidae), but extensive data for larger areas are still rare. Slovakia, with well-explored black fly fauna, was chosen to verify the reliability of DNA barcoding for species identification. The DNA barcoding region of the COI gene of 235 individuals of 25 black fly species from Slovakia was sequenced. Among them, 30 sequence clusters with assigned Barcode Index Numbers (BINs) were identified, and 5 of them were recorded for the first time. The average intraspecific genetic divergence was 0–3.24%, whereas the average interspecific divergence was 12.3–17.8%. Based on the barcode sequence, 14 species could be identified unambiguously, and 3 of them (Prosimulium latimucro, Simulium costatum, S. degrangei) are split into two or more barcode clusters. In eleven species, some degree of barcode sharing occurred, often combined with barcode splitting. The results showed hidden diversity as well as cases of shared barcode sequences among the studied species. Further investigation using other molecular markers is necessary due to the overlap of intraspecific and interspecific variability.

To date, a rather large set of both mathematical theories for species delimitation, based on single-locus genetic data, and their implementations as software products, has been accumulated. Comparison of the efficiencies of different delineation methods in the task of accumulating and analyzing data with reference to different taxa in different regions, is vital. The aim of this study was to compare the efficiency of fifteen single-locus species delimitation methods using the example of a fish species found in a single lake in European Russia (Lake Plescheyevo) with reference to other sequences of revealed taxa deposited in international databases. We analyzed 186 original COI sequences belonging to 24 haplotypes, and 101 other sequences previously deposited in GenBank and BOLD. Comparison of all 15 alternative taxonomies demonstrated that all methods adequately separate only the genera, while the number of delimited mOTUs differed from 16 (locMin) to 43 (HwM/CoMa). We can assume that the effectiveness of each method is correlated with the number of matches based on Ctax and MatchRatio criteria. The most comparable results were provided by bGMYC, mPTP, STACEY, KoT and ASAP and the most synchronous results were obtained from bGMYC, mPTP, STACEY and ASAP. We believe that these results are maximally realistic in the number of revealed mOTUs. A high genetic diversity, resulting in the existence of several mOTUs and phylogenetic lineages within many species, demonstrates the usefulness of the “polymorphic species” concept, which does not underestimate species richness and does not prevent the rational use and protection of biodiversity.

DNA barcoding is a powerful method for the identification of lichenized fungi groups for which the diversity is already well-represented in nucleotide databases, and an accurate, robust taxonomy has been established. However, the effectiveness of DNA barcoding for identification is expected to be limited for understudied taxa or regions. One such region is Antarctica, where, despite the importance of lichens and lichenized fungi identification, their genetic diversity is far from characterized. The aim of this exploratory study was to survey the lichenized fungi diversity of King George Island using a fungal barcode marker as an initial identification tool. Samples were collected unrestricted to specific taxa in coastal areas near Admiralty Bay. Most samples were identified using the barcode marker and verified up to the species or genus level with a high degree of similarity. A posterior morphological evaluation focused on samples with novel barcodes allowed for the identification of unknown Austrolecia, Buellia, and Lecidea s.l. species. These results contribute to better represent the lichenized fungi diversity in understudied regions such as Antarctica by increasing the richness of the nucleotide databases. Furthermore, the approach used in this study is valuable for exploratory surveys in understudied regions to guide taxonomic efforts towards species recognition and discovery.

Natural history collections are the physical repositories of our knowledge on species, the entities of biodiversity. Making this knowledge accessible to society – through, for example, digitisation or the construction of a validated, global DNA barcode library – is of crucial importance. To this end, we developed and streamlined a workflow for ‘museum harvesting’ of authoritatively identified Diptera specimens from the Smithsonian Institution’s National Museum of Natural History. Our detailed workflow includes both on-site and off-site processing through specimen selection, labelling, imaging, tissue sampling, databasing and DNA barcoding. This approach was tested by harvesting and DNA barcoding 941 voucher specimens, representing 32 families, 819 genera and 695 identified species collected from 100 countries. We recovered 867 sequences (> 0 base pairs) with a sequencing success of 88.8% (727 of 819 sequenced genera gained a barcode > 300 base pairs). While Sanger-based methods were more effective for recently-collected specimens, the methods employing next-generation sequencing recovered barcodes for specimens over a century old. The utility of the newly-generated reference barcodes is demonstrated by the subsequent taxonomic assignment of nearly 5000 specimen records in the Barcode of Life Data Systems.

Lansium domesticum is one of tropical plant. Genetic studies of tropical plant species occurred very slowly due to some obstacles. Provided fresh sample for isolation step was one of impediment. Fresh sample was important conditions to avoid impure DNA template. A simple and rapid technique was needed to get a pure DNA from storage sample. The aims of this study is to see the purity of the DNA isolation results from Lansium domesti-cum mature leaf from South Sumatra that have been stored for 2 years and to test whether the DNA isolation results can be used for barcoding analysis by amplification using PCR method with rbcL primer. There are 11 sample of Lansium domesticum were taken from 8 districts in South Sumatra. The sample was the mature leaves of Lanisum domesticum. The leaves were stored in a freezer at -20oC for two years. There was one sam-ple stored within 2 weeks as a comparison. The results of the study showed the isolation of DNA from storage and mature leaves of Lansium domesticum used Plant Genomic DNA Kit DP305 had a good quantity for 7 sample. 3 sample had low purity and 1 sample contaminated by RNA. Despite electrophoresis showed the isola-tion result not intact and compact, nevertheless the PCR results showed 11 samples could be amplified using rbcL primer. This result indicated the DNA isolation method suit for barcoding analysis using PCR method.

The analysis of DNA barcoding data to determine whether species are endemic, or non-endemic to specific regions in Southern Canada is difficult, but the existence of differences in summer and winter temperatures in the Canadian Arctic represent possible opportunities in methods of analysis that have not yet been undertaken. Species present in the Arctic are largely unique, but as climate change progresses the differences in climate that exist between Southern Canada and the Canadian Subarctic, and the Canadian Arctic are shrinking. As that change occurs, the detection of overlapping species range extents is likely possible using geospatial analysis and DNA barcodes.

Background Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods. Results With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80–90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes. Conclusions This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Determining the main pollinators and the factors that control their abundance is a prerequisite for improving the production of many crops, including the globally important cash crop cacao (Theobroma cacao L.). Yet, the identity and the breeding habitats of cacao pollinators remain controversial since they are very difficult to identify morphologically due to their small size. It is known that tiny midges are involved in cacao pollination, but their diversity and ecology remain obscure. Here, we combined the collection of flower visitors, the use of emergence traps, and DNA barcoding to assess the diversity of dipteran cacao flower visitors and link them to five candidate breeding habitats in an agroforestry system in central Nicaragua. We detected 59 lineages of dipteran flower visitors, including Cecidomyiidae, Ceratopogonidae, Chironomidae, and Sciaridae, suggesting that dipteran cacao pollinator diversity may be much higher than previously assumed. Five lineages could be genetically traced back to specific breeding habitats. We conclude that the presented approach can be a promising tool to reliably delineate potential pollinator species and their breeding habitats, which can inform management strategies to promote cacao pollination.

The geometrid moth genus Eumera Staudinger, 1892 consists of five yellow-orange-pinkish species distributed in the western Palearctic, with uncertain tribal classification within the geometrid subfamily Ennominae. In this study, we explored the phylogenetic position of the genus Eumera. Therefore, a concatenated dataset was analyzed, which includes one mitochondrial and up to ten protein-coding genetic markers per taxa. Moreover, we compared some external and internal morphological traits to other closely related genera. Our phylogenetic inference and comparative morphology suggested that Eumera should be included in the tribe Prosopolophini. In addition, a new species, Eumera rajaeii sp. nov. Wanke & Shirvani is described from southern Iran, and diagnosed by molecular data and morphological features. The distribution of the Iranian species is shown on a map. We illustrate external characters and male genitalia of three closely related Eumera species.

The species delimitation of the marine bivalve species complex Aequiyoldia eightsii in South America and Antarctica is complicated by mitochondrial heteroplasmy and amplification bias in molecular barcoding. In this study, we compare different data sources (mitochondrial cytochrome c oxidase subunit I (COI) sequences; nuclear and mitochondrial SNPs). Whilst all the data suggest that populations on either side of the Drake Passage belong to different species, the picture is less clear within Antarctic populations, which harbor three distinct mitochondrial lineages (p-dist ≈ 6%) that coexist in populations and in a subset of individuals with heteroplasmy. Standard barcoding procedures lead to amplification bias favoring either haplotype unpredictably and thus overestimate the species richness with high confidence. However, nuclear SNPs show no differentiation akin to the trans-Drake comparison, suggesting that the Antarctic populations represent a single species. Their distinct haplotypes likely evolved during periods of temporary allopatry, whereas recombination eroded similar differentiation patterns in the nuclear genome after secondary contact. Our study highlights the importance of using multiple data sources and careful quality control measures to avoid bias and increase the accuracy of molecular species delimitation. We recommend an active search for mitochondrial heteroplasmy and haplotype-specific primers for amplification in DNA-barcoding studies.

Most species of Megastigmus are considered important economic pests that grow in seeds, especially of conifers. Accurate identification of species is a crucial step for the biological research of parasitic pests and the further application of biological control. However, their large variety, small size, similar morphology and different growth and development stages have brought great challenges to taxonomic research. Traditional morphological identification often takes a long time and this requires us to seek a new method for rapid and accurate identification. Therefore, the better identification of Megastigmus urgently needs to be combined with molecular methods to help taxonomic development. Here, Megastigmus daduheensis sp. n. (Chalcidoidea: Megastigmidae) was identified, based on morphology and molecular markers, such as COI and Cytb. M. daduheensis sp. n. is distinct from other known species of the same genus in the morphology. The results of the molecular phylogenetic tree, similarity alignment and genetic distance indicate that the COI and Cytb sequences of M. daduheensis sp. n. are highly similar to M. sobinae and M. duclouxiana, but there are some genetic differences. The genetic distances of M. daduheensis sp. nov. with M. duclouxiana and M. sabinae were 0.34 and 0.33 and the percentages of shared base pairs were 76.3% and 76.8%, respectively. Both morphological and molecular data classified M. daduheensis sp. n. as a new species. The obtained COI and Cytb sequences of M. daduheensis sp. n. can be used as DNA barcodes, providing molecular data for rapid and accurate identification of this species in the future.

Natural hybridization can have a profound evolutionary impact, with consequences ranging from the extinction of rare taxa to the origin of new species. Natural hybridization is particularly common in plants, however our understanding of the general factors that promote or prevent hybridization are hampered by the highly variable outcomes in different lineages. Here, we quantify the influence of different predictors on hybrid formation across species from an entire flora. We combine estimates of hybridization with ecological attributes and a new species level phylogeny for over 1,100 UK flowering plant species. Our results show genetic factors, particularly parental genetic distance, as well as phylogenetic position and ploidy, are key determinants of hybrid formation, whereas many other factors such as range overlap and genus size explain much less variation in hybrid formation. Overall, intrinsic genetic factors shape the evolutionary and ecological consequences of natural hybridization across species in a flora.

Natural hybridization can have a profound evolutionary impact, with consequences ranging from the extinction of rare taxa to the origin of new species. Natural hybridization is particularly common in plants; however, our understanding of the general factors that promote or prevent hybridization is hampered by the highly variable outcomes in different lineages. Here, we quantify the influence of different predictors on hybrid formation across species from an entire flora. We combine estimates of hybridization with ecological attributes and a new species-level phylogeny for over 1,100 UK flowering plant species. Our results show that genetic factors, particularly parental genetic distance, as well as phylogenetic position and ploidy, are key determinants of hybrid formation, whereas many other factors such as range overlap and genus size explain much less variation in hybrid formation. Overall, intrinsic genetic factors shape the evolutionary and ecological consequences of natural hybridization across species in a flora.

Phylogenetic analysis is provided for the first time for 12 species of Palaearctic elfin butterflies, members of the previously recognized genera Ahlbergia Bryk, 1947, Cissatsuma Johnson, 1992, and Novosatsuma Johnson, 1992, based on the barcoding region of the mitochondrial cytochrome C oxidase subunit I gene (COI). Comparison of the COI barcodes revealed very low levels of genetic divergence between the species of the Palaearctic elfin butterflies and Callophrys Billberg, 1820 sensu stricto. COI-based phylogeny revealed that Palaearctic Callophrys and the Palaearctic elfin butterflies, except Cissatsuma, are polyphyletic. Four new sympatric species, namely, Callophrys (Ahlbergia) hmong sp. n., C. (A.) tay sp. n., Callophrys (Cissatsuma) devyatkini sp. n. and C. (A.) dao sp. n. are described from Ha Giang Province, North Vietnam, based on wing colouration, the morphologies of the male and female genitalia, and differences in COI sequences. Discovery of the new species expands the distribution range of the group towards the southeast, beyond the Palaearctic region.