Search Results (45)

Crete, a major Mediterranean biodiversity hotspot, hosts many local endemic, threatened and/or protected plant taxa (species and subspecies). Besides their ecological and conservation significance, these unique phytogenetic resources hold significant economic potential for sustainable utilization. Since DNA barcoding is critical for conservation, taxonomy, and plant-derived product authentication, we studied 15 local Cretan endemic taxa using three chloroplast DNA (cpDNA) regions (rbcL, trnL, trnH-psbA). A comparative analysis against GenBank (NCBI) records revealed significant new data: (i) the first genetic information for five taxa (Centaurea redempta subsp. redempta, Galium fruticosum, Micromeria hispida, Salix kaptarae, Teucrium cuneifolium); (ii) new marker-specific sequences for seven taxa (Helichrysum heldreichii, Scutellaria hirta, Sesleria doerfleri, Staehelina petiolata, Teucrium alpestre, Campanula pelviformis, Phlomis lanata); and (iii) novel genotypes of already represented markers for three species (Phlomis lanata, Scutellaria sieberi, Staehelina petiolata). Phylogenetic analyses were performed for all three molecular markers across selected members of Scutellaria section Scutellaria, Teucrium section Polium, and Campanula section Quinqueloculares. The overall results indicated that, amongst the studied species, the trnH-psbA marker is more suitable for species-level identification, whereas the rbcL and trnL markers were more helpful to genus-level identification within Lamiaceae and Campanulaceae. These results enrich the DNA barcoding reference library and form a concrete contribution towards the protection, conservation and traceability of Crete’s unique botanical heritage. Full article

Accurate sex identification in reptiles with genotypic sex determination is essential for breeding management, veterinary care and evolutionary research, yet commonly used methods are often invasive, stressful or unreliable. This study aimed to evaluate a dosage-based quantitative PCR approach for molecular sex determination in caenophidian snakes, using naturally shed epidermal skin as a non-invasive DNA source. Genomic DNA extracted from shed skin was analysed by qPCR targeting conserved Z-linked genes (ADARB2, ARMC4 and TANC2), together with autosomal and reference genes, to assess sex-specific differences in gene copy number. Sixteen caenophidian snake species were examined, including taxa for which molecular sexing data are currently scarce or unavailable. The autosomal control gene showed dosage ratios close to parity between sexes, supporting DNA quality and reference gene reliability; meanwhile, Z-linked markers generally exhibited reduced dosage in females relative to males, consistent with a ZZ/ZW sex determination system. These results demonstrate that dosage-based qPCR applied to shed epidermal skin provides a promising and non-invasive framework for molecular sex determination in caenophidian snakes, without compromising animal welfare. Full article

Wildlife trafficking poses a severe threat to global biodiversity and ecosystem stability, necessitating robust forensic tools for tracing the origins of illegally traded taxa. In this study, we developed a method of single-nucleotide polymorphism (SNP)-based molecular markers to enable precise geographical traceability of four animal species native to China: the Tibetan macaque (Macaca thibetana), brown eared pheasant (Crossoptilon mantchuricum), blue eared pheasant (Crossoptilon auritum), and Chinese pangolin (Manis pentadactyla). We studied these four species because their DNA is characterized by distinct population genetic structure, they are subjected to illegal trafficking, and given their diverse evolutionary histories, this allowed us to assess the general applicability of our forensic genetic framework in reducing wildlife crime. Based on whole-genome resequencing data from 26 Tibetan macaques, 51 eared pheasants and 42 Chinese pangolins, we performed population genetic analyses to elucidate their genetic structure and identify population-specific loci. The results indicated that all samples from these four species showed clear genetic differentiation and distinct clustering, allowing us to design primers to facilitate PCR-based traceability. We also assessed the utility of mitochondrial DNA (mtDNA) for tracing Tibetan macaques and both species of eared pheasants. We found that traceability accuracy using mtDNA was lower than when using SNPs. Our research offers a SNP-based traceability framework that accurately determines the geographical origin of wildlife samples to the genetic population level, and this provides a powerful tool for combating illegal trade and aiding conservation efforts. Full article

DNA metabarcoding data obtained by next generation sequencing (NGS) has been used to identify species in mixed biological samples, such as DNA from the gut content of invertebrates that feed on vertebrates (invertebrate-derived DNA, iDNA). This investigation employed DNA metabarcoding approach to determine vertebrate hosts of female phlebotomine sand flies, blood-feeding leishmaniasis vectors. We evaluated performance across three mitochondrial markers: a mammal-specific mini-barcode (16S rRNA), a pan-vertebrate mini-barcode (12S rRNA), and a standard CytB barcode region. Phlebotomine sand flies collections occurred in the Cacao Region of Southeastern Bahia, Brazil, an American Tegumentary Leishmaniasis (ATL) endemic zone. Our analysis examined iDNA from forty female specimens pooled in thirteen samples of seven sand fly species, including confirmed ATL vectors. Metabarcoding-derived operational taxonomic units (OTUs) underwent taxonomic assignment through comparison with GenBank NCBI^® reference databases. Results identified twenty vertebrate OTUs: primates (four OTUs), rodents (four), ungulates (five), marsupials (one), plus a domestic dog and a chicken. Notably, non-mammalian taxa, including reptiles (one OTU) and amphibians (three), were detected. The iDNA metabarcoding approach allowed us to accurately sample the diversity of phlebotomine sandflies’ bloodmeals in a single specimen of a non-engorged female sand fly with mixed feeding. Full article

Background/Objectives: While morphological similarity and incomplete specimens pose a challenge to the precise identification of Coelogyne orchids, accurate species and genus assignment is essential for conservation and CITES enforcement. This study evaluated the efficacy of five DNA barcode regions—rbcL, matK, trnH-psbA, atpF-atpH, and ITS2—and their combinations for species- and genus-level discrimination within the genus Coelogyne, aiming to develop a rapid and simple diagnostic tool for use by customs officers and trade inspectors. This is the first comprehensive comparative analysis of these five barcode regions specifically within Coelogyne, a genus underrepresented in molecular identification studies, and the first to propose multi-locus combinations for potential practical use. This study identified DNA barcode regions with high resolution and reliability, providing a solid basis for practical identification kits. Such tools will enhance CITES enforcement by enabling rapid detection of Coelogyne species in trade, directly supporting their conservation and contributing to the reduction in illegal orchid trade. Methods: Using a CTAB protocol, genomic DNA was extracted from leaf samples belonging to 19 Coelogyne species. Sanger sequencing was performed after PCR amplification using published primer sets for every barcode region. Sequences were modified in BioEdit, and BLASTn (accessed 15 June 2025) was used to compare them to GenBank (NCBI Nucleotide). Amplification efficiency was calculated per locus. Species and genus identification success rates were determined by the congruence of top BLAST hits with morphologically pre-identified taxa. Multi-barcode combinations (matK + rbcL, ITS2 + matK, matK + trnH-psbA, rbcL + trnH-psbA, and matK + rbcL + trnH-psbA) were also assessed. Results: With rbcL, atpF-atpH, and ITS2 yielding ≤11%, the highest single-locus species identification rates were for trnH-psbA (21%) and matK (16%). Among single-locus barcodes, matK showed the highest performance, with 84% genus assignment. ITS2 reached 27%, but genus-level resolution remained limited for the rbcL, trnH-psbA and atpF-atpH barcodes. Multi-barcode approaches maintained species resolution: matK + rbcL + trnH-psbA, matK + rbcL, and matK + trnH-psbA correctly identified 16% of species and achieved 74–79% genus assignment. Conclusions: No single locus achieves robust species discrimination in Coelogyne, but trnH-psbA, matK and atpF-atpH provide the best single-marker performance. Using the matK locus alone, in combination with either trnH-psbA or rbcL, or all three together ensures consistent genus-level identification and significantly improves taxonomic resolution. This study introduces a novel multi-locus barcode strategy tailored to Coelogyne, offering a practical solution for identification and enforcement. While promising, this approach represents a potential application that requires further validation before routine implementation. Full article

Understanding the mechanisms that assemble diverse forest communities is a central goal in ecology. Phylogenetic analyses based on DNA barcodes have advanced this field, but their use of sequences evolving at constant rates may not capture adaptations to specific environmental drivers. Light is a critical factor shaping forest structure, particularly in the vertical dimension. This study introduces a novel phylogenetic approach using the blue-light receptor gene, cryptochrome (Cry), which is directly involved in plant light perception and adaptation. We reconstructed a Cry-based phylogeny for 96 tree species in a 20 ha subtropical forest dynamics plot and analyzed community structure using the net relatedness index (NRI) and nearest taxon index (NTI) across horizontal habitats, successional stages, and vertical canopy layers. Compared to traditional DNA barcoding, the Cry phylogeny revealed distinct patterns, showing consistent phylogenetic structure across different habitats—a finding indicative of convergent evolution in light-sensing systems. Furthermore, the Cry-based analysis demonstrated a stronger and more consistent signal in the forest’s vertical structure, with significant phylogenetic clustering in upper canopy layers, directly linking light adaptation to community stratification. Over time, both NRI and NTI values increased, suggesting succession leads to greater phylogenetic overdispersion and highlighting an increased role for environmental filtering among closely related taxa. Our results validate Cry as a powerful functional gene marker for phylogenetics, providing unique insights into how light environment filters species and shapes the vertical assembly of forest communities. Full article

Cyst nematodes of the genus Heterodera are important plant-parasitic nematodes that cause significant crop losses worldwide but are often overlooked due to their non-specific symptoms and complex biology. This study assessed Heterodera diversity in Portugal using an integrative molecular approach based on four genetic markers (mtCOI, 18S rDNA, ITS, and 28S rDNA). Five valid species were identified: Heterodera cruciferae, H. mani, H. schachtii, H. trifolii, and H. zeae, with H. mani reported for the first time in the country. A distinct taxon from Coimbra (central Portugal) may represent a new or unsequenced species, highlighting gaps in reference datasets. Among the markers, mtCOI was the most effective, though some taxa remained unresolved. These results reinforce the value of multi-marker approaches, contribute with new sequences, and improve diagnostic capability for nematode management. Full article

Resolving the eukaryotic tree of life (eToL) remains a fundamental challenge in biology. Much of eukaryotic phylogenetic diversity is occupied by unicellular microbial eukaryotes (i.e., protists). Among these, the phylogenetic positions of a significant number of lineages remain unresolved due to limited data and ambiguous traits. To address this issue, we introduce the term “PUPAs” (protists with uncertain phylogenetic affiliations) to collectively describe these lineages, instead of using vague or inconsistent labels, such as incertae sedis or orphan taxa. Historically, protists were classified based solely on morphological features, and many with divergent cell structures were left unplaced in the eToL. With the advent of sequence-based approaches, the phylogenetic affiliations of some PUPAs have been clarified using molecular markers, such as small subunit ribosomal DNA. The combination of technological progress and continuous efforts to cultivate diverse protists, including PUPAs and novel protists, now enables phylogenetic analyses based on hundreds of proteins, providing their concrete placements in the eToL. For example, these advances have led to the discovery of new deep-branching lineages (e.g., Hemimastigophora), the resolution of relationships among major groups (e.g., Microheliella, which linked Cryptista and Archaeplastida), and insights into evolutionary innovations within specific clades (e.g., Glissandra). In this review, we summarize current consensus in eukaryotic phylogeny and highlight recent findings on PUPAs whose phylogenetic affiliations have been clarified. We also discuss a few lineages for which the phylogenetic homes remain unsettled, the evolutionary implications of these discoveries, and the remaining challenges in resolving the complete eToL. Full article

The Maghreb region of North Africa, located at the intersection of the Palaearctic and Afrotropical zones, is a biodiversity hotspot for terrestrial and freshwater taxa, including the freshwater crab of genus Potamon Savigny, 1816. Recent molecular studies have suggested the presence of two distinct Potamon species in the region: Potamon algeriense Bott, 1967, and an as-yet undescribed taxon, Potamon sp. However, comprehensive data on their distribution, genetic structure, and conservation status are still lacking. In the present study, we integrate new field collections from Algeria and Morocco (2021–2023) with molecular analyses of mitochondrial (COI, ND1) and nuclear (28S rDNA) markers to assess species boundaries and genetic diversity within Potamon across the Maghreb. Phylogenetic reconstructions based on Maximum Likelihood and Bayesian Inference consistently support the presence of two well-differentiated Potamon lineages in the region, corresponding to P. algeriense in western and central Maghreb, and Potamon sp. in eastern Algeria and Tunisia. While Potamon sp. exhibits low intra-specific genetic variation, P. algeriense displays a deeply structured mitochondrial lineage composition, forming four geographically coherent subclades, each corresponding to distinct hydrological regions. In light of this, it would be advisable to revise the IUCN assessment to include both species and updated information on their distribution. Full article

The number of Fritillaria species native to the Alps has long been debated, and observational biases due to the short flowering periods and the scattered distributions of endemic Fritillaria populations along the mountain range have probably made the task of botanists more complicated. Moreover, previous phylogenetic studies in Fritillaria have considered alpine taxa only marginally. To test species boundaries within the F. tubaeformis species complex and to study their phylogenetic relationships, intra- and inter-specific genetic variability of sixteen samples belonging to four Fritillaria species was carried out in different localities of the Maritime and Ligurian Alps, with extensions to the rest of the Alpine arc. The combined use of five plastid DNA markers (matK, ndhF, rpl16, rpoC1, and petA-psbJ) and nrITS showed that F. tubaeformis and F. burnatii are phylogenetically independent taxa, fully confirming morphological and morphometric divergences and, that F. burnatii is not related phylogenetically to the central European F. meleagris. Our phylogenetic study also supports the separation of F. tubaeformis from F. moggridgei, pointing to environment/ecological constraints or reproductive barriers as possible causes of their distinct evolutionary status. Our analysis also showed that the mountain endemic F. involucrata is not closely related to F. tubaeformis, contrasting with previous studies. The phylogenetic analysis of the nrITS region supports a close relationship between F. burnatii and F. moggridgei, but with low statistical support. Full article

The taxonomic identification of plant species is traditionally based on morphological traits, the use of which may create difficulties in cases of close-related species showing great morphological variability. In such cases, the use of DNA markers for species identification and delimitation can be of great help. Himantoglossum W.D.J.Koch (Orchidaceae) is a genus with notable morphological variability, comprising the clade hircinum-caprinum (Himantoglossum s.s.) with nine taxa, from which H. jankae, H. hircinum, H. montis-tauri, H. caprinum and H. samariense have being reported in Greece. However, a previous morphological study of Himantoglossum s.s. from all over Greece could not verify the presence of these reported species, but of only one highly diverse taxon throughout the country. Here, we studied the genetic variation and differentiation of Himantoglossum s.s. populations from the entire distribution of the genus in Greece employing ISSR markers, to further elucidate the taxonomic status of Himantoglossum s.s. in Greece. High genetic variation was revealed, both in the populations of the “core” distribution and in the peripheral/marginal ones, pointing to their evolutionary potential. This variation is mainly attributed to differences within the populations and, to a lesser extent, among them. No differentiation of the populations proposed to belong to a different taxon was found and no species-specific markers were identified that may discriminate the above populations from the rest. In addition, two cpDNA and one nDNA fragments (accD, psbA-trnH and ITS2, respectively) were sequenced in a number of individuals representative of the whole dataset. All three fragments were conserved, showing restricted polymorphism and having no correlation to the populations or to the taxa of Himantoglossum s.s. in Greece. Overall, the high genetic variation of the populations of Himantoglossum s.s. in Greece, especially of the peripheral/marginal ones, is a valuable asset towards their conservation. Full article

Background: While intratumoral microbiota have been identified in various cancers, their presence and clinical significance in lacrimal gland tumors remain largely unexplored. This study investigates the existence, composition, and potential clinical significance of intratumoral bacteria in lacrimal gland tumors. Methods: High-throughput 16S rDNA sequencing was performed on tumor DNA extracted from 89 paraffin-embedded tissues from patients with lacrimal gland tumors. Diversity analysis and LEfSe differential analysis were conducted to identify tumor-type-specific bacterial taxa. LASSO regression and the Cox proportional hazards models were used to analyze the relationship between intratumoral microbiota and prognosis. Results: Significant differences in the β diversity of intratumoral microbiota were observed across adenoid cystic carcinoma (ACC), carcinoma ex pleomorphic adenoma (CXPA), pleomorphic adenoma (PA), and IgG4-related disease (IgG4-RD) patients. After FDR correction, Garicola, Prevotella, Polaribacter, and Helicobacter were notably enriched in the tumors of ACC, CXPA, PA, and IgG4-RD patients, respectively. Importantly, patients with malignant lacrimal gland tumors who experienced relapse, distant metastasis, or death had significantly higher α diversity within their tumors. Furthermore, specific genera, such as Roseburia and Alloprevotella, were particularly associated with poorer prognosis in patients with malignant lacrimal gland tumors. Conclusions: This study provides a comprehensive analysis of microbial profiles in lacrimal gland tumors, highlighting distinct microbial characteristics across tumor types. Our findings suggest that intratumoral bacterial diversity and specific genera may serve as potential prognostic markers for malignant lacrimal gland tumors. Full article

Research in metagenomics and metaproteomics can reveal how microbiological interactions in fermented foods contribute to their health benefits. This study examined three types of fermented vegetables: a standard formulation, a probiotic formulation with Lacticaseibacillus rhamnosus GG, and a polyphenol formulation with vitexin from Mung bean seed coat. Measurements were taken at day 0 (after 36 h of fermentation at room temperature) and after 15 days. We applied 16S rRNA sequencing to evaluate microbial diversity and utilized LC-MS/MS to investigate the proteomic profiles of specific genera (Lactobacillus and Weissella) and species (Lacticaseibacillus rhamnosus and Levilactobacillus brevis) of lactic acid bacteria (LAB). All of these taxa demonstrated significant relative abundance between 0 and 15 days of fermentation in our metagenomic analysis. Our findings from principal component analysis and clustering analysis categorically distinguished protein expression patterns at various stages of fermentation. By comparing samples from day 0 to day 15, we identified proteins associated with DNA replication and repair mechanisms, including transcription elongation factor GreA, tRNA pseudouridine synthase B, and helicases. We also observed their roles in protein synthesis, which encompasses oxidoreductases and aspartokinase. Furthermore, we identified strong correlations of specific proteins across the three formulations with antioxidant markers. In conclusion, the results of this study decisively enhance our understanding of the role of the proteins related to specific LAB in fermented foods, highlighting their potential to improve texture, flavor, nutritional quality, and health benefits. Full article

The Danube Clouded Yellow (Colias myrmidone) has experienced one of the most dramatic declines among European butterflies. To estimate genetic diversity in the last population in Poland that has survived in the Knyszyn Forest (KF), we analyzed mitochondrial (COI) and nuclear (EF-1α) polymorphisms in individuals sampled in 2014 and 2022. The results were compared with genetic data obtained in 2014 from a recently extirpated nearby population (Czerwony Bór, CB). Because mtDNA polymorphisms in insects can be modulated by endosymbionts, the samples were screened for Wolbachia. The polymorphism of EF-1α indicated that diversity was gradually decreasing. The KF experienced rapid demographic processes, manifested by a significant change in allele frequency. The small differentiation in nuclear markers between the KF and CB in 2014 suggests that the regional population used to be genetically uniform. Four COI haplotypes that were identified in this study probably belong to two different haplogroups. Wolbachia was detected only in individuals with one specific haplotype, and the prevalence was female-biased, suggesting the induction of two reproductive manipulations. The most common COI haplotype found in Poland was the same as that reported from other parts of Europe, not only for C. myrmidone but also C. caucasica. These results allow us to question the distinctiveness of each taxa. Full article

Background/Objectives: Sium serra is distributed in Korea, China, and Japan. It was first identified as the genus Pimpinella and then reclassified as Sium by Kitagawa. Some Sium species are used as herbal medicine and are often confused with the similar form Ligusticum sinense. In this study, we analyzed the cp genome of S. serra and conducted comparative analyses with the cp genomes of related taxa. Methods: We extracted gDNA from fresh leaves and sequenced it using Illumina HiSeq2500. For the chloroplast genome assembly, de novo assembly was performed using Velvet v1.2.07. For the annotation, GeSeq and NCBI BLASTN were used. Afterwards, related taxa were analyzed using programs such as DnaSP and MISA. Results: S. serra was excluded from the study on the chloroplast (cp) genome in Sium because it was classified as Pimpinella in China. Therefore, this study aimed to analyze the cp genome of S. serra for the first time and its location within the genus Sium. The complete cp genome of S. serra was 154,755 bp in length, including a pair of inverted repeats, each 26,255 bp, a large single-copy region of 84,581 bp, and a small single-copy region of 17,664 bp. The cp genome comprised 79 protein-coding, 30 tRNA, and 4 rRNA genes. Furthermore, six regions of high nucleotide diversity were identified in the genus Sium. In the genus Sium, 1630 repeats that can serve as markers were also identified. Eight protein-coding genes with high K_A/K_S values were under positive selection in the Sium. Our phylogenetic analyses suggest that S. serra was positioned with high bootstrap support within the Sium of the tribe Oenantheae, specifically in the southern Palearctic subclade. Conclusions: In this study, the S. serra chloroplast genome was sequenced and assembled. The genus Sium formed a monophyletic group; however, as not all the Sium species were included in this study, further research is necessary. This study can serve as foundational data not only for Sium but also for the tribe Oenantheae. Full article