Reliable Species Identification Research Articles

Pest categorisation of non-EU Scolytinae on non-coniferous hosts.

The EFSA Panel on Plant Health performed a group pest categorisation for the EU territory of non-EU Scolytinae (Coleoptera: Curculionidae) on non-coniferous hosts, which total 6495 known species. Most species attack apparently healthy, weakened or dead trees, either feeding on the phloem ('bark beetles' subgroup) or on fungi inoculated into the sapwood ('ambrosia beetles' subgroup). Smaller subgroups feed and reproduce in seeds and fruits, or in herbaceous plants. Some species are polygynous, the males initiate a gallery or a chamber on or in a new host and attract females. Others are monogamous, and the females initiate the new galleries. Many species respond to primary volatile attractants emitted by the hosts, and some produce aggregation pheromones that attract conspecifics of both sexes. The species attacking living hosts are often associated with fungi that contribute to weakening the host defences and provide nutrients to the insects. Some are inbreeding; the males in the offspring mate with their sisters and rarely leave their natal tree. The larvae of all species develop and pupate within their hosts. Based on catalogues and other published data, a database was constructed providing information on hosts, feeding and reproductive habits, geographic distribution and the Köppen-Geiger climate types in countries where species occur. The Scolytinae were screened to exclude species in the following categories: (i) 708 species attacking conifers; (ii) 127 species present in at least four EU Member States and (iii) 440 species occurring in areas with climatic conditions not occurring in the EU. Among the remaining 5220 species, 88 species known for their mobility, occupying at least two landmasses separated by geographical barriers and some of which had impact levels documented in literature, were extracted. They were grouped into four subcategories: (i) 12 species with high impact on plant health; (ii) 16 species with low or doubtful impact; (iii) 48 species with no impact; (iv) 12 species with no impact and which had never been recorded as 'introduced' in the consulted catalogues but occurring on at least two landmasses. All 88 species could enter the EU with wood or wood products, or with plants for planting, and could establish because host plants are available, and climate is suitable in parts of the EU. Control measures to inhibit introduction are available. There is considerable uncertainty regarding the potential impact of many species. Methods for the reliable identification of many species are lacking. For some species of non-EU Scolytinae on non-coniferous hosts, all criteria assessed by EFSA for consideration as potential quarantine pest are met. Nevertheless, the Panel was not able to develop a method to discriminate confidently between species that clearly meet the criteria for potential quarantine pest status and those that do not.

Unique findings of <i>Phoma</i>-like fungi associated with soybean

Ascochyta leaf blight of soybean is a widespread disease caused by several closely related Phoma-like species, this disease often leads to significant crop losses. Among Phoma-like species from Didymellaceae family, the most frequently associated with symptomatic soybean tissues are species of the genera Boeremia and Didymella. Currently reliable species identification in Didymellaceae relies on polyphasic approach based on consolidated species concept and combined molecular phylogenetic, micromorphological and cultural features. At least three loci are commonly used for reconstruction of the molecular phylogeny of Didymellaceae: internal transcribed spacer (ITS) of the ribosomal DNA, partial RNA-polymerase II gene (rpb2), and β-tubulin (tub2). As a result of long-term phytosanitary monitoring of soybean crops, soybean leaves with symptoms of Ascochyta blight were collected from major soybean producing areas of Russia. From surface sterilized plant tissues more than 100 isolates of Phoma-like fungi were obtained and stored in the collection of pure cultures of the Laboratory of Mycology and Phytopathology (MF, All-Russian Institute of Plant Protection). Most of them, as a result of multilocus phylogenetic analysis, were identified as Boeremia and Didymella species. Eight isolates were identified as species of other genera, suspected to be rare findings. The aim of this study was to identify these eight isolates based on multilocus phylogenetic analysis, as well micromorphological, cultural, and pathogenicity data. Multilocus phylogenetic analysis has resulted in identification of all eight isolates to species level. Single isolate from the Ryazan region was Neoascochyta graminicola. Three other from three different districts of the Amur region were Remotididymella capsici. Two isolates from the Primorskiy territory and Amur region were Stagonosporopsis heliopsidis. Another two from two districts of the Amur region were S. stuijvenbergii. Pathogenicity tests have resulted in conclusion, that all studied isolates were not pathogenic for soybean leaves. Probably, these Phoma-like species are associate with soybean as saprophytes or endophytes. For all these Phoma-like species Glycine max was detected as substrate for the first time. Neoascochyta graminicola is widespread in Europe in association with Poaceae plants. There are only two findings of Remotididymella capsici in the world, both from leaves of Capsicum annuum. First finding was made in the former USSR in 1977 and was identified based on only morphological features. Second findings was collected in the Fiji and verified with multilocus phylogenetic analysis. Stagonosporopsis heliopsidis isolates were revealed in the USA, Canada, Netherlands and Russia and this fungus was believed to be specific for Asteraceae plants. Isolates of Stagonosporopsis stuijvenbergii are known only from soil in the Netherlands. Thus, such species as Neoascochyta graminicola and Stagonosporopsis stuijvenbergii were revealed in the Russia for the first time. Studied Remotididymella capsici isolates were first confirmed findings of this fungus in Russia. Additionally to detailed phylogenetic data, the manuscript is supplement with a detailed description of the cultural and micromorphological features of all species.

Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan.

The majority of myxosporean species (Cnidaria: Myxozoa) of the genera Myxobolus (35 species), Henneguya (8 species), and Myxidium (9 species) from freshwater or brackish fish in Japan were recorded more than 30 years ago (accumulatively 81.1% [43/53]). The re-discovery and molecular-genetic characterization of these species is a current research priority. During our myxosporean survey in Japanese freshwater fish, we detected three species that had never been recorded in Japan, but in the Russian Far East (Sakhalin Island, and Maritime Province): Myxobolus tribolodonus sp. n., forming cysts in the gills of Tribolodon sachalinensis (syn. M. marinus sensu Aseeva, 2000; M. marinus sensu Sokolov et Frolova, 2015, recorded from the gills of Pseudaspius (syn. Tribolodon) spp.); Henneguya pungitii Achmerov, 1953, forming cysts in the subcutis of external skin and buccal submucosa of Pungitius sinensis; and Myxidium salvelini Konovalov et Shulman, 1966, in the urinary bladder of Oncorhynchus masou ishikawae. These new isolates were characterized by integrated taxonomic approaches, i.e., myxospore morphology and molecular-genetic characterization of the small subunit ribosomal RNA gene (SSU rDNA). These new isolates were phylogenetically differentiated from any species whose SSU rDNA sequences were deposited in the DNA databases, and concurrently compared with recorded species based on classical morphological criteria. All three species were differentiated from myxosporeans previously recorded in Japan, indicating new distribution records out of the Russian Far East. For reliable species identification, accumulation of at least SSU rDNA sequences of known species worldwide is critically important.

New and interesting records of rare caddisflies (Trichoptera, Insecta) from Slovakia with comments on their ecology and conservation status

The distribution and diversity of insects are crucial conservation issues. The order Trichoptera is comprised of numerous endangered species, but the rarity of some makes it challenging to determine their conservation priority. In recent decades, data based on caddisfly larvae have become more frequent in limnological research in Slovakia. Our research, focusing mainly on adult caddisflies, has led to more reliable and accurate identification of many species that cannot be identified with certainty in the larval stage.We combined several entomological methods, including sweeping, Malaise traps, and UV light attraction, to increase the likelihood of catching rare species. The text lists and discusses the discoveries of caddisfly species that are particularly important for faunistics or biodiversity in Slovakia, as well as in the Central European context. Enoicyla reichenbachii (Kolenati, 1848), Limnephilus incisus Curtis, 1834, Ptilocolepus granulatus (Pictet, 1834) and Oecetis testacea (Curtis, 1834) were recorded for the first time in Slovakia. The rare species Hagenella clathrata (Kolenati, 1848) and Adicella balcanica Botosaneanu and Novák, 1965, previously found in Slovakia, were also recorded. Additionally, a new locality of Adicella syriaca Ulmer, 1907 indicates a further northward shift of this Mediterranean species. We further discussed the faunistically important records of Beraeamyia hrabei Mayer, 1937, Allotrichia pallicornis (Eaton, 1873), Ceraclea riparia (Albarda, 1874), Grammotaulius nitidus (Müller, 1764) and Limnephilus fuscicornis Rambur, 1842 from Slovakia. The conservation status of each species is proposed and discussed on the basis of our faunistic records, in accordance with the forthcoming update of the Slovak Red Book.

Fusion of Laser-Induced Breakdown Spectroscopy and Raman Spectroscopy for Mineral Identification Based on Machine Learning.

Rapid and reliable identification of mineral species is a challenging but crucial task with promising application prospects in mineralogy, metallurgy, and geology. Spectroscopic techniques such as laser-induced breakdown spectroscopy (LIBS) and Raman spectroscopy (RS) efficiently capture the elemental composition and structural information of minerals, making them a potential tool for in situ and real-time analysis of minerals. This study introduces an integrated LIBS-RS system and the fusion of LIBS and RS spectra coupled with machine learning to classify six different types of natural mineral. In order to visualize the separability of different mineral species clearly, the spectral data were projected into low-dimensional space through t-distributed stochastic neighbor embedding (t-SNE). Additionally, the Fisher score (FS) was used to identify important variables that contribute to the data classification, and the corresponding chemical elements and molecular bonds were then interpreted. The between-minerals difference in the feature spectral intensity of LIBS and RS variables could also be observed. After the minerals spectra were pre-processed, the relationship between spectral intensity and the mineral category was modeled using machine learning methods, including partial least squares-discriminant analysis (PLS-DA) and kernel extreme learning machine (K-ELM). The results show that K-ELM and PLS-DA based on the fusion LIBS-RS data achieved the highest accuracy of 98.4%. These findings demonstrate the feasibility of the integrated LIBS-RS system combined with machine learning for the fast and reliable classification of minerals.

Loop‐mediated isothermal amplification (LAMP) assays for Aedes and Culex species and evaluation of a simple DNA preparation method for field application

Loop‐mediated isothermal amplification (<scp>LAMP</scp>) assays for <i>Aedes</i> and <i>Culex</i> species and evaluation of a simple <scp>DNA</scp> preparation method for field application

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species

BackgroundMosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species.MethodsSampling was carried out in three biogeographical regions of Croatia over six years (2017–2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs.ResultsA total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna.ConclusionWe present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.Graphical

Färgvariation hos vattenpiplärka Anthus spinoletta i vinterdräkt

This study of colour variation on Water Pipit Anthus spinoletta in winter plumage, is based on photos of 93 individuals from southern Sweden. Using moulting criteria, the birds were aged from the photographs, and the presence or absence of five specific plumage characteristics were recorded. Twelve individuals in this study confirm the hypothesis that adult Water Pipits show a distinct combination of plumage traits during winter. However, the presence of these plumage traits varied in younger birds in their first winter plumage. Among the 81 individuals classified as first winter plumage, 23 (28%) displayed all “adult characteristics”, 32 (40%) one to four of the characteristics, while 26 (32%) lacked all “adult characteristics”. The latter group thus lacked a gray nape/crown, whitish supercilium, and lighter brown colouration of the mantle/scapulars. Ever since the first Swedish records in the mid-1990s, there have been ongoing discussions regarding species-specific characters in Water Pipits. The present quantification of colour variation in different age groups complements previous criteria for a reliable species identification of Water Pipits in winter plumage.

Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple.

Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer-probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.

A Genetic Tool to Identify Predators Responsible for Livestock Attacks in South America and Recommendations for Human-Wildlife Conflict Mitigation.

Livestock predation induces global human-wildlife conflict, triggering the retaliatory killing of large carnivores. Although domestic dogs (Canis familiaris) contribute to livestock depredation, blame primarily falls on wild predators. Dogs can also transmit pathogens between wildlife, domestic animals, and humans. Therefore, the presence of free-ranging dogs can have negative consequences for biodiversity conservation, smallholder economy, food supply, and public health, four of the United Nations' Sustainable Developed Goals (SDGs) for 2030. In Ecuador, where livestock sustains rural households, retaliatory poaching threatens Andean bear (Tremarctos ornatus), jaguar (Panthera onca), and puma (Puma concolor) populations. However, the role of dogs in these incidents remains underexplored. The present study evaluates the possibility of reliable molecular identification of predatory species from DNA traces in bite wounds. Our results revealed the presence of dog saliva on four out of six livestock carcasses presumably attacked by wild predators. These findings highlight the importance of rectifying misinformation about large carnivores in Ecuador and the need to control dog populations. We recommend that local administrations incorporate DNA analysis into livestock predation events to examine how common the problem is, and to use the analysis to develop conflict mitigation strategies which are essential for the conservation of large carnivores.

Application of biomolecular techniques on tsetse fly puparia for species identification at larvipostion sites.

Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

Identification of closely related species in Aspergillus through Analysis of Whole-Genome.

The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus Aspergillus and developed a rapid detection method. First, reliable whole genome data for 77 Aspergillus species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus flavus, and Aspergillus terreus) and three well-known species (A. flavus, Aspergillus parasiticus, and Aspergillus oryzae) within the Aspergillus section. Flavi and AGE have provided species-level-specific targets for 77 species within the genus Aspergillus. Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/μl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Species identification of adult ixodid ticks by Raman spectroscopy of their feces

BackgroundTicks and tick-borne diseases pose significant challenges to cattle production, thus the species identification of ticks and knowledge on their presence, abundance, and dispersal are necessary for the development of effective control measures. The standard method of inspection for the presence of ticks is the visual and physical examination of restrained animals, but the limitations of human sight and touch can allow larval, nymphal, and unfed adult ticks to remain undetected due to their small size and site of attachment. However, Raman spectroscopy, an analytical tool widely used in agriculture and other sectors, shows promise for the identification of tick species in infested cattle. Raman spectroscopy is a non-invasive and efficient method that employs the interaction between molecules and light for the identification of the molecular constituents of specimens.MethodsRaman spectroscopy was employed to analyze the structure and composition of tick feces deposited on host skin and hair during blood-feeding. Feces of 12 species from a total of five genera and one subgenus of ixodid ticks were examined. Spectral data were subjected to partial least squares discriminant analysis, a machine-learning model. We also used Raman spectroscopy and the same analytical procedures to compare and evaluate feces of the horn fly Haematobia irritans after it fed on cattle.ResultsFive genera and one sub-genus at overall true prediction rates ranging from 92.3 to 100% were identified from the Raman spectroscopy data of the tick feces. At the species level, Dermacentor albipictus, Dermacentor andersoni and Dermacentor variabilis at overall true prediction rates of 100, 99.3 and 100%, respectively, were identified. There were distinct differences between horn fly and tick feces with respect to blood and guanine vibrational frequencies. The overall true prediction rate for the separation of tick and horn fly feces was 98%.ConclusionsOur findings highlight the utility of Raman spectroscopy for the reliable identification of tick species from their feces, and its potential application for the identification of ticks from infested cattle in the field.Graphical

PARSID: a Python script for automatic analysis of local BLAST results for a rapid molecular taxonomic identification

ObjectiveA reliable taxonomic identification of species from molecular samples is the first step for many studies. For researchers unfamiliar with programming, running a BLAST analysis, filtering, and organizing results for hundreds of sequences through the BLAST web interface can be difficult. Additionally, sequences deposited in GenBank can have outdated taxonomic identification. The use of reliable Reference Sequences Library (RSL) containing accurate taxonomically-identified sequences facilitates this task. Pending the availability of a RSL with the user, we developed a tool that automates the molecular taxonomic identification of sequences.ResultsWe developed PARSID, a Python script running through the command-line that automates the routine workflow of blasting an input sequence file against the user’s RSL, and retrieves the matches with the highest percentage of identity in five steps. PARSID accepts cut-off parameters and supplementary information in a.csv file for filtering the results. The final output is visualized in a spreadsheet. We tested its functioning using 10 input sequences simulating different situations of the molecular taxonomic identification of sequences against an example RSL containing 25 sequences. Step-by-step instructions and test files are publicly available at https://github.com/kokinide/PARSID.git.

DNA barcoding-based molecular profiling of Bougainvillea, Dianthus, and Plumeria using matK locus

DNA barcoding, a robust tool for species identification, holds significant promise for enhancing authenticity and quality control in herbal and Ayurvedic medicines. The traditional methods, often time-consuming and impractical for industrial-scale applications, are complemented by DNA barcoding, which offers efficient and reliable plant species identification. However, the identification of the subspecies or variants within species is a difficult task and has challenges due to higher sequence similarity for marker genes and correct identification of variants. This research presents a comprehensive methodology for DNA barcoding in three variants of common medicinal plants, namely, Bougainvillea, Dianthus, and Plumeria, using the maturase K (matK) as a molecular marker. The sequenced matK regions with 500–700 bp size have a variability of approximately 1% to 2.7% among inter and intra-species variants, facilitating the easy identification of the species through high-quality barcodes. The sequence and phylogenetic analysis revealed that our sequences were clustered with the matK genes of closely related species. The two-dimensional barcodes generated for the nine inter and intra-species variants can be scanned easily. Our findings underscore the potential of DNA barcoding as a powerful tool for the identification and authentication of inter and intra-species variants over conventional means.

Genetic variations and phylogenetic relationship of genus Uromastyx from Punjab Pakistan.

During the present study, specimens were collected from selected sites of Cholistan desert and Kalabagh Game Reserve, Punjab province, Pakistan. Each captured specimen was tagged with voucher number and morphometric measurements were taken. The average snout to vent length was 172.559±1.40 mm and average weight was 92.1±1.30 g. The DNA of Uromastyx hardwickii was amplified and sequenced using 16S rRNA primer set. The obtained DNA sequence has shown reliable and clear species identification. After trimming ambiguous bases, the obtained 16S rRNA fragment was 520 bp while 16S rRNA fragments aligned with closely matched sequence from NCBI comprised of 510 bp. Closely matched sequences of genus Uromastyx were retrieved from NCBI in blast searches. Neighbour-joining tree of genus Uromastyx was constructed based on p-distance using MEGA X. The mean intraspecific variation was 0.095±0.01 while intraspecific variation was ranging from 0-1%. Similarly, interspecific variation of Uromastyx hardwikii with Saara asmussi, Uromastyx alfredschmidti, Uromastyx geyri, Uromastyx thomasi, Uromastyx alfredschmidti was 0-12%, 0-19%, 0-19%, 0-20%, 12-19% respectively. The newly produced DNA was submitted to NCBI and accession number was obtained (MW052563.1). Results of current study provided information about the molecular and morphological identification of Genus Uromastyx. In our recommendation, comprehensive molecular based identification of Pakistan's reptiles is required to report any new or subspecies from country.

Diaporthe species on palms – integrative taxonomic approach for species boundaries delimitation in the genus Diaporthe, with the description of D. pygmaeae sp. nov.

The application of traditional morphological and ecological species concepts to closely related, asexual fungal taxa is challenging due to the lack of distinctive morphological characters and frequent cosmopolitan and plurivorous behaviour. As a result, multilocus sequence analysis (MLSA) has become a powerful and widely used tool to recognise and delimit independent evolutionary lineages (IEL) in fungi. However, MLSA can mask discordances in individual gene trees and lead to misinterpretation of speciation events. This phenomenon has been extensively documented in Diaporthe, and species identifications in this genus remains an ongoing challenge. However, the accurate delimitation of Diaporthe species is critical as the genus encompasses several cosmopolitan pathogens that cause serious diseases on many economically important plant hosts. In this regard, following a survey of palm leaf spotting fungi in Lisbon, Portugal, Diaporthe species occurring on Arecaceae hosts were used as a case study to implement an integrative taxonomic approach for a reliable species identification in the genus. Molecular analyses based on the genealogical concordance phylogenetic species recognition (GCPSR) and DNA-based species delimitation methods revealed that speciation events in the genus have been highly overestimated. Most IEL identified by the GCPSR were also recognised by Poisson tree processes (PTP) coalescent-based methods, which indicated that phylogenetic lineages in Diaporthe are likely influenced by incomplete lineage sorting (ILS) and reticulation events. Furthermore, the recognition of genetic recombination signals and the evaluation of genetic variability based on sequence polymorphisms reinforced these hypotheses. New clues towards the intraspecific variation in the common loci used for phylogenetic inference of Diaporthe species are discussed. These results demonstrate that intraspecific variability has often been used as an indicator to introduce new species in Diaporthe, which has led to a proliferation of species names in the genus. Based on these data, 53 species are reduced to synonymy with 18 existing Diaporthe species, and a new species, D. pygmaeae, is introduced. Thirteen new plant host-fungus associations are reported, all of which represent new host family records for Arecaceae. This study has recognised and resolved a total of 14 valid Diaporthe species associated with Arecaceae hosts worldwide, some of which are associated with disease symptoms. This illustrates the need for more systematic research to examine the complex of Diaporthe taxa associated with palms and determine their potential pathogenicity. By implementing a more rational framework for future studies on species delimitation in Diaporthe, this study provides a solid foundation to stabilise the taxonomy of species in the genus. Guidelines for species recognition, definition and identification in Diaporthe are included.

Taxonomic reappraisal of the European fauna of the bark beetle genus Cryphalus (Coleoptera, Curculionidae, Scolytinae).

Species in the genus Cryphalus are small and notoriously difficult to identify. Even among the relatively well studied European species, erroneous identifications are evident from literature and in museum collections. These misidentifications relate to the small size and similar appearance of Cryphalus species but they are also a product of insufficient diagnostic characters. This is especially unfortunate since some European species are considered pests. Based on the study of more than 1000 specimens and a thorough literature review, robust morphological and molecular evidence supporting all five hitherto recognised native species of Cryphalus in Europe is provided. A key for the reliable identification of these repetition species including new diagnostic characters recognised for the first time, including those from male genitalia, has been constructed. Each native species is provided with a detailed morphological description and their economic significance, distribution, and ecology discussed. Significant genetic variability is observed between certain clusters that should be further explored in a broader geographic context. Lastly, the need for a taxonomic revision of the genus Cryphalus for the entire Palearctic region due to the presence of many similar looking species which are often confused, thus distorting the knowledge of each species is highlighted.