Enhancing metabarcoding efficiency and ecological insights through integrated taxonomy and DNA reference barcoding: A case study on beach meiofauna.

Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5-fold increase in sequence annotation and a doubling of species-level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell-shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems.

A review of the genus Zygota (Hymenoptera, Diapriidae) in Germany with taxonomic notes on this genus and its distinction from Pantoclis.

This study provides a comprehensive overview of the genus Zygota Förster combining DNA barcoding and current morphology. Nineteen species of Zygota were found throughout Germany, including the newly described species Zygotawalli sp. nov. First species records for Germany are: Zygotabalteata Macek, 1997; Z.comitans Macek, 1997; Z.spinosipes (Kieffer, 1908); Z.sordida Macek, 1997; Z.angularis Macek, 1997 and Z.vigil Nixon, 1957. We also clarify diagnoses for the two related genera, Pantoclis Förster and Zygota to designate the boundaries of the Zygota genus and propose new synonymies: Zygotacaligula Buhl, 1997 is a junior synonym of Z.congener (Zetterstedt, 1840); Z.reticulata Kozlov, 1978 is a junior synonym of Z.ruficornis (Curtis, 1831). Thirteen species of Zygota sensu Nixon (1957) are transferred to the genus Pantoclis with the following new combinations proposed: Zygotabrevinervis (Kieffer, 1908) (= Pantoclisbrevinervis (Kieffer, 1909), comb. nov.); Z.brevipennis (Kieffer, 1908) (= P.brevipennis (Kieffer, 1908), comb. nov.); Z.caecutiens (Kieffer, 1908) (= P.caecutiens (Kieffer, 1908), comb. nov.); Z.cursor (Kieffer, 1908) (= P.cursor (Kieffer, 1908), comb. nov.); Z.fossulata (Thomson, 1858) (=P.fossulata (Thomson, 1858), comb. nov.); Z.fuscata (Thomson, 1858) (= P.fuscata (Thomson, 1858), comb. nov.); Z.hemiptera (Thomson, 1858) (= P.hemiptera (Thomson, 1858), comb. nov.); Z.microtoma (Kieffer, 1909) (= P.microtoma (Kieffer, 1909), comb. nov.); Z.soluta (Kieffer, 1907) (= P.soluta (Kieffer, 1907), comb. nov.); Z.striata (Kieffer, 1909) (= P.striata (Kieffer, 1909), comb. nov.); Z.subaptera (Thomson, 1858) (= P.subaptera (Thomson, 1858), comb. nov.); Z.sulciventris (Kieffer, 1909) (= P.sulciventris (Kieffer, 1909), comb. nov.), and Z.unicolor (Kieffer, 1908) (= P.unicolor (Kieffer, 1908), comb. nov.).

Corrigendum: Ex vivo modulation of intact tumor fragments with anti-PD-1 and anti-CTLA-4 influences the expansion and specificity of tumor-infiltrating lymphocytes.

[This corrects the article DOI: 10.3389/fimmu.2023.1180997.].

Characterization and evaluation of nine Cannabis sativa chloroplast SNP markers for crop type determination and biogeographical origin on European samples.

Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.

Cupiennius spiders (Trechaleidae) from southern Mexico: DNA barcoding, venomics, and biological effect.

Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.

Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies.

Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between pre-clinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine.

Unveiled species diversity of moss-feeding mites (Stigmaeidae: Eustigmaeus): a research on their distribution, habitat, and host plant use in Japan.

The genus Eustigmaeus Berlese, 1910 represents the unique phytophagous group within the superfamily Raphignathoidea. Four species within this genus have been known to inhabit mosses and feed on them as larvae, nymphs, and adults. However, the interactions with mosses have remained poorly understood. In order to reveal the diversity and host-plant use of the moss-feeding species, we conducted an extensive field study in Japan. This study revealed an array of moss-feeding species inhabiting various moss species, with 10 morphologically distinctive species newly documented in Japan. Through DNA barcoding based on cytochrome c oxidase subunit I (COI) sequences, these morphospecies were recovered as distinct entities. Notably, the host-plant use of four species was elucidated. Among these, Eustigmaeus sp. 9 exhibited polyphagy, while three species (Eustigmaeus spp. 1-3) demonstrated varying degrees of host specificity, each using moss species from the Hypnales, Philonotis, and Dicranidae, respectively. While a few moss-feeding species were frequently found in the same geographic area, more than one species rarely co-occurred within the same moss colonies. Eustigmaeus offers a unique study system, with its diverse moss-feeding species and indications of specific host plant use. Consequently, the moss-feeding Eustigmaeus serves as a valuable model for exploring the macroevolutionary patterns underlying diversification in moss-feeding arthropods.

Description of Trichophoromyia jariensis, a new species of phlebotomine sand fly (Diptera: Psychodidae) from the eastern Amazon.

A new sand fly species, Trichophoromyia jariensis n. sp. Cavalcante, Rodrigues, & Galati, from the state of Amapá, Brazil, is described based on both male and female morphology and cytochrome c oxidase subunit I DNA barcodes. The DNA barcoding analysis clearly associated males and females of this new species.

Successful development of molecular diagnostic technology combining mini-barcoding and high-resolution melting for traditional Chinese medicine agarwood species based on single-nucleotide polymorphism in the chloroplast genome.

Agarwood is a valuable traditional medicine and fragrance. The production process is a typical injury-induced defense response. Currently, there are approximately 22 known species in the genus Aquilaria Lam., all of which can produce agarwood, whereas there are only two legal species of traditional Chinese medicinal agarwood, Aquilaria sinensis (Lour.) Spreng. and Aquilaria agallocha (Lour.) Roxb. The Taiwan herbal Pharmacopoeia of China stipulates that the medicinal agarwood species are A. sinensis and its relatives in the same genus. Moreover, there are five species of agarwood available for clinical medicinal use in Japan, including A. agallocha and A. sinensis, which are often confused with each other or used in a mixed way in the trade process. Therefore, accurate identification of traditional Chinese medicinal agarwood species is important to ensure the authenticity of traditional medicines and to guide the safety of clinical medication. In this study, 59 specific single-nucleotide polymorphism loci were screened and obtained from the chloroplast genomes of 12 species of the genus Aquilaria Lam. We established an identification method for traditional Chinese medicinal agarwood using mini-barcoding combined with high-resolution melting (HRM) and designed and validated 10 pairs of primers from the psbM-trnD, psbA, rps16, petN, ndhE-psaC, rps4, atpE, ycf1, rps15-trnN, and matK regions. The amplification products were all less than 200 bp, with a high success rate of amplification. The method was applied to successfully identify traditional Chinese medicinal agarwood species from commercial agarwood samples. Overall, the sensitivity of this method was sufficient to detect 1% of adulterants in medicinal agarwood products, proving that mini-barcoding HRM is a powerful and flexible tool. This method can be used as a fast and effective high-throughput method for authenticity testing of traditional Chinese medicinal agarwood and its raw materials containing agarwood-containing proprietary Chinese medicines and is recommended for industrial applications.

DNA barcoding combined with high-resolution melting analysis to discriminate rhubarb species and its traditional Chinese patent medicines.

Introduction: Rhubarb is a frequently used and beneficial traditional Chinese medicine. Wild resources of these plants are constantly being depleted, meaning that rhubarb products have been subjected to an unparalleled level of adulteration. Consequentially, reliable technology is urgently required to verify the authenticity of rhubarb raw materials and commercial botanical drugs. Methods: In this study, the barcode-DNA high-resolution melting (Bar-HRM) method was applied to characterize 63 rhubarb samples (five Polygonaceae species: Rheum tanguticum, Rh. palmatum, Rh. officinale, Rumex japonicus and Ru. sp.) and distinguish the rhubarb contents of 24 traditional Chinese patent medicine (TCPM) samples. Three markers, namely ITS2, rbcL and psbA-trnH, were tested to assess the candidate DNA barcodes for their effectiveness in distinguishing rhubarb from its adulterants. A segment from ITS2 was selected as the most suitable mini-barcode to identify the botanical drug rhubarb in TCPMs. Then, rhubarbs and TCPM samples were subjected to HRM analysis based on the ITS2 barcode. Results: Among the tested barcoding loci, ITS2 displayed abundant sites of variation and was effective in identifying Polygonaceae species and their botanical origins. HRM analysis based on the ITS2 mini-barcode region successfully distinguished the authenticity of five Polygonaceae species and eight batches of TCPMs. Of the 18 TCPM samples, 66.7 % (12 samples) were identified as containing Rh. tanguticum or Rh. officinale. However, 33.3 % were shown to consist of adulterants. Conclusions: These results demonstrated that DNA barcoding combined with HRM is a specific, suitable and powerful approach for identifying rhubarb species and TCPMs, which is crucial to guaranteeing the security of medicinal plants being traded internationally.

Convergent technologies to tackle challenges of modern food authentication.

The authentication process involves all the supply chain stakeholders, and it is also adopted to verify food quality and safety. Food authentication tools are an essential part of traceability systems as they provide information on the credibility of origin, species/variety identity, geographical provenance, production entity. Moreover, these systems are useful to evaluate the effect of transformation processes, conservation strategies and the reliability of packaging and distribution flows on food quality and safety. In this manuscript, we identified the innovative characteristics of food authentication systems to respond to market challenges, such as the simplification, the high sensitivity, and the non-destructive ability during authentication procedures. We also discussed the potential of the current identification systems based on molecular markers (chemical, biochemical, genetic) and the effectiveness of new technologies with reference to the miniaturized systems offered by nanotechnologies, and computer vision systems linked to artificial intelligence processes. This overview emphasizes the importance of convergent technologies in food authentication, to support molecular markers with the technological innovation offered by emerging technologies derived from biotechnologies and informatics. The potential of these strategies was evaluated on real examples of high-value food products. Technological innovation can therefore strengthen the system of molecular markers to meet the current market needs; however, food production processes are in profound evolution. The food 3D-printing and the introduction of new raw materials open new challenges for food authentication and this will require both an update of the current regulatory framework, as well as the development and adoption of new analytical systems.

Species identification of livefood flightless fly (Torinido-shoujoubae) through DNA barcoding.

Torinido-shoujoubae, as it is called in Japanese, is a flightless Drosophila sp. that is sold commercially in Japan. This Drosophila sp. is often used as feeds for model organisms such as reptiles and spiders. There is no scientific name provided for the fruit fly that is known as Torinido-shoujoubae, as well as any historical background or data behind this species. There has been a previous study that was conducted through morphological characteristics analysis of the body as well as the male copulatory organ and has been estimated as Drosophila hydei. The objective of this study was to determine the species of this unidentified fly known as Torinido-shoujoubae based on a molecular evidence with a DNA barcoding. Samples were purchased from four separate suppliers to examine whether there are any differences between them. COI regions were amplified using PCR and the sequenced results were aligned against two databases, NCBI and BOLD. Torinido-shoujoubae samples provided from all suppliers were confirmed to be D. hydei.

Forensic species identification: practical guide for animal and plant DNA analysis.

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

A phylogenetic study of Micareamelaeniza and similar-looking species (Pilocarpaceae) unveils hidden diversity and clarifies species boundaries and reproduction modes.

Micarea (Ascomycota, Pilocarpaceae) is a large cosmopolitan genus of crustose lichens. We investigated molecular systematics and taxonomy of the poorly known Micareamelaeniza group focussing on M.melaeniza, M.nigella and M.osloensis. A total of 54 new sequences were generated and using Bayesian and maximum likelihood analysis of two markers (nuITS and mtSSU), we discovered two previously unrecognized phylogenetic lineages, one of which is described here as Micareaeurasiatica Kantelinen & G. Thor, sp. nov., morphologically characterized by pycnidia that are sessile to emergent, cylindrically shaped, with greenish-black K+ olive green, wall pigmentation and containing large mesoconidia up to 6 µm in length. The species is known from Japan and Finland. In addition, we show that the reproduction biology of M.osloensis has been poorly understood and that the species often occurs as an anamorph with stipitate pycnidia. We present a species synopsis and notes on pigments. Our research supports previous results of asexuality being an important reproductive strategy of species growing on dead wood.

DNA barcoding is currently unreliable for species identification in most crayfishes.

DNA barcoding is commonly used for species identification. Despite this, there has not been a comprehensive assessment of the utility of DNA barcoding in crayfishes (Decapoda: Astacidea). Here we examined the extent to which local barcoding gaps (used for species identification) and global barcoding gaps (used for species discovery) exist among crayfishes, and whether global gaps met a previously suggested 10× threshold (mean interspecific difference being 10× larger than mean intra specific difference). We examined barcoding gaps using publicly available mitochondrial COI sequence data from the National Center for Biotechnology Information's nucleotide database. We created two versions of the COI datasets used for downstream analyses: one focused on the number of unique haplotypes (N H) per species, and another that focused on total number of sequences (N S; i.e., including redundant haplotypes) per species. A total of 81 species were included, with 58 species and five genera from the family Cambaridae and 23 species from three genera from the family Parastacidae. Local barcoding gaps were present in only 30 species (20 Cambaridae and 10 Parastacidae species). We detected global barcoding gaps in only four genera (Cambarus, Cherax, Euastacus, and Tenuibranchiurus), which were all below (4.2× to 5.2×) the previously suggested 10× threshold. We propose that a ~5× threshold would be a more appropriate working hypothesis for species discovery. While the N H and N S datasets yielded largely similar results, there were some discrepant inferences. To understand why some species lacked a local barcoding gap, we performed species delimitation analyses for each genus using the N H dataset. These results suggest that current taxonomy in crayfishes may be inadequate for the majority of examined species, and that even species with local barcoding gaps present may be in need of taxonomic revisions. Currently, the utility of DNA barcoding for species identification and discovery in crayfish is quite limited, and caution should be exercised when mitochondrial-based approaches are used in place of taxonomic expertise. Assessment of the evidence for local and global barcoding gaps is important for understanding the reliability of molecular species identification and discovery, but outcomes are dependent on the current state of taxonomy. As this improves (e.g., via resolving species complexes, possibly elevating some subspecies to the species-level status, and redressing specimen misidentifications in natural history and other collections), so too will the utility of DNA barcoding.

Can consumers avoid mislabelling? Genetic species identification provides recommendations for shrimp/prawn products.

BACKGROUND: Crustaceans of the superfamily Penaeoidea (e.g., shrimps and prawns) are among the most commercially available aquatic products worldwide. However, there are few studies regarding not only the presence but also the characteristics of mislabelling in these food products. Such information would be helpful for consumers in order to avoid the typical problems associated with mislabelling (e.g., health and economic issues). For this reason, this work considers Penaeoidea mislabelling by comparing different products (frozen, fresh, boiled), and sources (hypermarkets, supermarkets and fishmongers) from Spain (Europe). RESULTS: A total of 94 samples from 55 different products were collected, representing 19 different species from 13 genera. Mitochondrial DNA (COI gene) was amplified, revealing mislabelling in almost 30% of supermarket products and almost exclusively found in frozen samples (95% of the total) regardless of its price. In addition, products from the Pacific Ocean seem to be particularly susceptible to mislabelling. CONCLUSIONS: All in all, recommendations for the consumer in order to avoid mislabelling of prawns include purchasing them fresh from fishmongers; aquaculture products must not be avoided. This study represents, to our knowledge, the first attempt to provide recommendations to consumers based on DNA analyses in order to avoid mislabelling in food products. Further research is therefore required to provide such recommendations in different food products, particularly those that are processed, packaged and/or frozen. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

DNA barcoding reveals a taxonomic fraud: Note on validity of Propomacrusmuramotoae (Coleoptera, Scarabaeidae).

Until the early 2000s, the genus Propomacrus was known to comprise two species, occurring in the Eastern Mediterranean and Southeast China. The discovery of Propomacrusmuramotoae Fujioka in Tibet and subsequently in Bhutan and Nepal, might play a crucial role in bridging the geographical distribution gap of the Euchirini tribe between the Mediterranean and Central China, offering profound insights into its evolution and biogeography. However, all specimens, including the holotype specimen, were sourced from a single insect vendor, with no further specimens found or catalogued in museum collections thereafter. During our examination of a P.muramotoae specimen from a private collection in South Korea, we found its COI gene sequence to be identical to that of P.bimucronatus (Pallas) from Turkey, a species known for its wide distribution and genetic variability across regional populations. This overlap in genetic identity raised significant doubts, further compounded by our detection of deliberate modifications in essential diagnostic features during morphological examination. All three specimens we examined showed crude modifications, including staining and artificial grinding. Despite our inability to access the P.muramotoae type specimens for direct examination-a challenge we attempted to overcome through various means-it is evident that significant fraudulent tampering has occurred with the P.muramotoae specimens. Therefore, a new synonymy is proposed: Propomacrusbimucronatus Pallas, 1781 = P.muramotoae Fujioka, 2007 (syn. nov.). We also advocate for a straightforward verification of the type specimen through molecular analysis of the COI barcode region and morphological re-examination under a microscope for those who have access to the type specimens.

Description of two species of the orb-weaver spider genus Argiope Audouin, 1826 (Araneae, Araneidae) from Xizang, China.

Background: The spider genus Argiope Audouin, 1826, comprises 88 species worldwide, including 23 species occurring in China. Two Argiope species were collected by the spider survey on Yarlung Zangbo Grand Canyon National Nature Reserve, Xizang, southwest China, conducted in 2023. New information: Two species of the orb-weaver spider genus Argiope from Xizang, China are described, including a new species, A.beibeng Mi & Wang, sp. nov. (♂♀) and a known species, A.caesarea Thorell, 1897 (♂♀). The unknown male of A.caesarea is described for the first time.

Second world record for Barathronus roulei Nielsen, 2019 (Ophidiiformes, Bythitidae), from the Porcupine Bank (Northeast Atlantic).

Barathronus is a genus of blind cusk eels comprising 11 valid species. In this paper, we report the second specimen ever documented of Barathronus roulei (Bythitidae) obtained from the Porcupine Bank by R.V. Vizconde de Eza using a bottom trawl at a depth of 1349 m. Morphological description and illustrations, including a radiograph, are provided. In addition, three new sequences corresponding to three different genes, cytochrome c oxidase subunit I (COI)-DNA barcoding, 16S ribosomal RNA (16S), and recombination activating protein 1 (RAG1), have been added to the molecular repositories, representing the first sequences for the species.

Metabarcoding analysis provides insight into the link between prey and plant intake in a large alpine cat carnivore, the snow leopard.

Species of the family Felidae are thought to be obligate carnivores. However, detection of plants in their faeces raises questions about the role of plants in their diet. This is particularly true for the snow leopard (Panthera uncia). Our study aimed to comprehensively identify the prey and plants consumed by snow leopards. We applied DNA metabarcoding methods on 90 faecal samples of snow leopards collected in Kyrgyzstan, employing one vertebrate and four plant markers. We found that argali (Ovis ammon) was detected only from male snow leopards. Myricaraia sp. was the most consumed among 77 plant operational taxonomic units found in snow leopard samples. It frequently appeared in samples lacking any prey animal DNA, indicating that snow leopards might have consumed this plant especially when their digestive tracts were empty. We also observed differences in the patterns of plant consumption between male and female snow leopards. Our comprehensive overview of prey and plants detected in the faeces of snow leopards and other sympatric mammals will help in formulating hypotheses and guiding future research to understand the adaptive significance of plant-eating behaviour in felids. This knowledge supports the enhancement of their captive environments and the conservation planning of their natural habitats.