Standard Barcode Research Articles

Organellar genome evolution in Ribes L.: hotspots of mutation, sequence transfer, codon usage, and phylogenetic context

Ribes genus from the Grossulariaceae family, have been used in the food industry and for medicinal purposes for years. Despite the valuable medicinal values and the widespread occurrence of Ribes, many species remain understudied. One of these is the alpine currant Ribes alpinum L., which occurs natively in Europe. The study aimed to thoroughly understand and characterize the organelle genomes of R. alpinum, conduct their comparative analysis with available data for other representatives of Grossulariaceae and, in the case of the chloroplast genome, also with Saxifraga insolens Irmsch., a representative of the closely related Saxifragaceae family. The analysis of plastome interspecific variation revealed several hotspots of variation which could be useful as potential phylogenetic markers or specific DNA barcodes. The phenomenon of sequence transfer between the chloroplast and mitochondrial genomes and the relative synonymous codon usage between the plastome and mitogenome were also analyzed. A phylogenetic inference based on the chloroplast genome sequence questioned earlier reports of a close relationship of R. alpinum to R. rubrum and confirmed its affiliation with an early-diverged phylogenetic line together with R. fasciculatum. Analysis of hotspots of variation in chloroplast genome allowed us to indicate petN-psbM and trnS-trnG as high-utility barcode sequences, competitive with standard barcodes and the ycf1 gene.

Sympatric diversity pattern driven by the secondary contact of two deeply divergent lineages of the soybean pod borer Leguminivora glycinivorella.

The soybean pod borer, Leguminivora glycinivorella (Matsumura), is an important tortricid pest species widely distributed in most parts of China and its adjacent regions. Here, we analyzed the genetic diversity and population differentiation of L. glycinivorella using diverse genetic information including the standard cox1 barcode sequences, mitochondrial genomes (mitogenomes), and single-nucleotide polymorphisms (SNPs) from genotyping-by-sequencing. Based on a comprehensive sampling (including adults or larvae of L. glycinivorella newly collected at 22 of the total 30 localities examined) that covers most of the known distribution range of this pest, analyses of 543 cox1 barcode sequences and 60 mitogenomes revealed that the traditionally recognized and widely distributed L. glycinivorella contains two sympatric and widely distributed genetic lineages (A and B) that were estimated to have diverged ∼1.14 million years ago during the middle Pleistocene. Moreover, low but statistically significant correlations were recognized between genetic differentiation and geographic or environmental distances, indicating the existence of local adaptation to some extent. Based on SNPs, phylogenetic inference, principal component analysis, fixation index, and admixture analysis all confirm the two divergent sympatric lineages. Compared with the stable demographic history of Lineage B, the expansion of Lineage A had possibly made the secondary contact of the two lineages probable, and this process may be driven by the climate fluctuation during the late Pleistocene as revealed by ecological niche modeling.

DNA barcoding: An effective molecular tool for species identification, molecular authentication and phylogeny studies in plant science research

DNA barcoding is a technique for identifying specimens using brief, standardised DNA segments. In a variety of fields, including phylogeny, ecology, population genetics and biodiversity, DNA barcoding has become a successful method for precisely distinguishing species. The method is straightforward, efficient in both time and money and accurate. The key to successful DNA barcoding is choosing the right DNA marker. Since the idea of a quick approach for species identification was first put up in 2003, the scientific community has been keen to realise the potential of DNA barcodes. Cytochrome c oxidase, I (COI) region of the mitochondrial genome is mostly recognised as a standard barcoding region in animals. Later, rbcL + matK pairing, with a 70 % discriminating efficiency, was suggested by the Plant Working Group (PWG) of the Consortium for the Barcoding of Life (CBOL) as the standard barcode in plants. Three conditions must be met for a gene region to be an efficient DNA barcode: it must have sufficient species-level genetic divergence and variability, it must have conserved flanking regions for the widest taxonomic use and for generating universal PCR primers and it should be long enough to facilitate current capability for sequencing and extracting DNA. Different combinations of plastid coding, non-coding and nuclear markers are utilised as supplemental markers to boost the degree of plant species differentiation. The reliability of different barcodes in distinguishing species varies among different groups of plants. As DNA barcoding approaches its twentieth anniversary, technologies are still being developed that make use of this resource, which is constantly expanding in a variety of biological disciplines. Plant DNA barcoding, which became a scientific advance during the last ten years, is frequently employed as a taxonomical aid in identifying species. It is a way of choosing genetic loci that identifies and distinguishes an organism's membership from specific species, variations or even intervarieties. It varies from molecular phylogeny, which identifies an unknown sample from an existing classification rather than identifying patterns of association.

Performance of DNA metabarcoding, standard barcoding and morphological approaches in the identification of insect biodiversity.

For two decades, DNA barcoding and, more recently, DNA metabarcoding have been used for molecular species identification and estimating biodiversity. Despite their growing use, few studies have systematically evaluated these methods. This study aims to evaluate the efficacy of barcoding methods in identifying species and estimating biodiversity, by assessing their consistency with traditional morphological identification and evaluating how assignment consistency is influenced by taxonomic group, sequence similarity thresholds and geographic distance. We first analysed 951 insect specimens across three taxonomic groups: butterflies, bumblebees and parasitic wasps, using both morphological taxonomy and single-specimen COI DNA barcoding. An additional 25,047 butterfly specimens were identified by COI DNA metabarcoding. Finally, we performed a systematic review of 99 studies to assess average consistency between insect species identity assigned via morphology and COI barcoding and to examine the distribution of research effort. Species assignment consistency was influenced by taxonomic group, sequence similarity thresholds and geographic distance. An average assignment consistency of 49% was found across taxonomic groups, with parasitic wasps displaying lower consistency due to taxonomic impediment. The number of missing matches doubled with a 100% sequence similarity threshold and COI intraspecific variation increased with geographic distance. Metabarcoding results aligned well with morphological biodiversity estimates and a strong positive correlation between sequence reads and species abundance was found. The systematic review revealed an 89% average consistency and also indicated taxonomic and geographic biases in research effort. Together, our findings demonstrate that while problems persist, barcoding approaches offer robust alternatives to traditional taxonomy for biodiversity assessment.

Applicability and perspectives for DNA barcoding of soil invertebrates

Belowground invertebrate communities are dominated by species-rich and very small microarthropods that require long handling times and high taxonomic expertise for species determination. Molecular based methods like metabarcoding circumvent the morphological determination process by assigning taxa bioinformatically based on sequence information. The potential to analyse diverse and cryptic communities in short time at high taxonomic resolution is promising. However, metabarcoding studies revealed that taxonomic assignment below family-level in Collembola (Hexapoda) and Oribatida (Acariformes) is difficult and often fails. These are the most abundant and species-rich soil-living microarthropods, and the application of molecular-based, automated species determination would be most beneficial in these taxa. In this study, we analysed the presence of a barcoding gap in the standard barcoding gene cytochrome oxidase I (COI) in Collembola and Oribatida. The barcoding gap describes a significant difference between intra- and interspecific genetic distances among taxa and is essential for bioinformatic taxa assignment. We collected COI sequences of Collembola and Oribatida from BOLD and NCBI and focused on species with a wide geographic sampling to capture the range of their intraspecific variance. Our results show that intra- and interspecific genetic distances in COI overlapped in most species, impeding accurate assignment. When a barcoding gap was present, it exceeded the standard threshold of 3% intraspecific distances and also differed between species. Automatic specimen assignments also showed that most species comprised of multiple genetic lineages that caused ambiguous taxon assignments in distance-based methods. Character-based taxonomic assignment using phylogenetic trees and monophyletic clades as criteria worked for some species of Oribatida but failed completely for Collembola. Notably, parthenogenetic species showed lower genetic variance in COI and more accurate species assignment than sexual species. The different patterns in genetic diversity among species suggest that the different degrees of genetic variance result from deep evolutionary distances. This indicates that a single genetic threshold, or a single standard gene, will probably not be sufficient for the molecular species identification of many Collembola and Oribatida taxa. Our results also show that haplotype diversity in some of the investigated taxa was not even nearly covered, but coverage was better for Collembola than for Oribatida. Additional use of secondary barcoding genes and long-read sequencing of marker genes can improve metabarcoding studies. We also recommend the construction of pan-genomes and pan-barcodes of species lacking a barcoding gap. This will allow both to identify species boundaries, and to cover the full range of variability in the marker genes, making molecular identification also possible for species with highly diverse barcode sequences.

Comparative chloroplast genome and species discrimination analyses in Aralia section Aralia (Araliaceae)

Aralia section Aralia is a subset of Araliaceae; it constitutes a small lineage that has been utilized in East Asian traditional medicines for centuries. Identifying species within this section can be challenging due to the complex interspecific morphological variations and the prevalence of hybridization. Accurate species identification is crucial for the application of these medicinal plants. In this study, we evaluated the effectiveness of 11 distinct barcode systems for distinguishing species of Aralia sect. Aralia. Comparative analyses of the chloroplast genomes suggested that the plastome length, gene content and order, as well as the expansion/contraction of the inverted repeat regions, are highly conserved. Species discrimination analysis revealed that complete plastomes and their partitioned data sets possessed considerably high discriminatory power (90.91%), which was equivalent to that of standard barcodes. In contrast, nuclear ribosomal DNA sequences demonstrated a marked improvement (90.91%) over nuclear ribosomal internal transcribed spacer (72.73%) in species discrimination. Evidently, nuclear markers did not perform better than plastid markers. However, a combination of barcode from both nuclear and chloroplast genomes can accurately identify all species for Aralia sect. Aralia. We therefore call for developing multilocus markers from different genomes with different inheritance for species discrimination in plant groups.

Testing plastomes and nuclear ribosomal DNA sequences as the next-generation DNA barcodes for species identification and phylogenetic analysis in Acer

BackgroundAcer is a taxonomically intractable and speciose genus that contains over 150 species. It is challenging to distinguish Acer species only by morphological method due to their abundant variations. Plastome and nuclear ribosomal DNA (nrDNA) sequences are recommended as powerful next-generation DNA barcodes for species discrimination. However, their efficacies were still poorly studied. The current study will evaluate the application of plastome and nrDNA in species identification and perform phylogenetic analyses for Acer.ResultBased on a collection of 83 individuals representing 55 species (c. 55% of Chinese species) from 13 sections, our barcoding analyses demonstrated that plastomes exhibited the highest (90.47%) species discriminatory power among all plastid DNA markers, such as the standard plastid barcodes matK + rbcL + trnH-psbA (61.90%) and ycf1 (76.19%). And the nrDNA (80.95%) revealed higher species resolution than ITS (71.43%). Acer plastomes show abundant interspecific variations, however, species identification failure may be due to the incomplete lineage sorting (ILS) and chloroplast capture resulting from hybridization. We found that the usage of nrDNA contributed to identifying those species that were unidentified by plastomes, implying its capability to some extent to mitigate the impact of hybridization and ILS on species discrimination. However, combining plastome and nrDNA is not recommended given the cytonuclear conflict caused by potential hybridization. Our phylogenetic analysis covering 19 sections (95% sections of Acer) and 128 species (over 80% species of this genus) revealed pervasive inter- and intra-section cytonuclear discordances, hinting that hybridization has played an important role in the evolution of Acer.ConclusionPlastomes and nrDNA can significantly improve the species resolution in Acer. Our phylogenetic analysis uncovered the scope and depth of cytonuclear conflict in Acer, providing important insights into its evolution.

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

Deep learning image analysis for filamentous fungi taxonomic classification: Dealing with small datasets with class imbalance and hierarchical grouping.

Deep learning applications in taxonomic classification for animals and plants from images have become popular, while those for microorganisms are still lagging behind. Our study investigated the potential of deep learning for the taxonomic classification of hundreds of filamentous fungi from colony images, which is typically a task that requires specialized knowledge. We isolated soil fungi, annotated their taxonomy using standard molecular barcode techniques, and took images of the fungal colonies grown in petri dishes (n = 606). We applied a convolutional neural network with multiple training approaches and model architectures to deal with some common issues in ecological datasets: small amounts of data, class imbalance, and hierarchically structured grouping. Model performance was overall low, mainly due to the relatively small dataset, class imbalance, and the high morphological plasticity exhibited by fungal colonies. However, our approach indicates that morphological features like color, patchiness, and colony extension rate could be used for the recognition of fungal colonies at higher taxonomic ranks (i.e. phylum, class, and order). Model explanation implies that image recognition characters appear at different positions within the colony (e.g. outer or inner hyphae) depending on the taxonomic resolution. Our study suggests the potential of deep learning applications for a better understanding of the taxonomy and ecology of filamentous fungi amenable to axenic culturing. Meanwhile, our study also highlights some technical challenges in deep learning image analysis in ecology, highlighting that the domain of applicability of these methods needs to be carefully considered.

Development of advanced PCR-based methods for accurate identification and authentication of commercial shrimp products

Shrimp is among the most economically valuable and susceptible seafood species to food fraud worldwide. The identities of 40 commercial shrimp products sold in South Korean markets were assessed using the standard barcoding method, revealing a significant 70% incidence of mislabeling. Based on these findings, three distinct PCR methods were developed to differentiate between eight predominant species (Penaeus vannamei, Pandalus borealis, Palaemon gravieri, Leptochela gracilis, Penaeus monodon, Pleoticus muelleri, Metapenaeopsis dalei, and Euphausia pacifica). Both conventional and real-time PCR (RT-PCR) showed specificity ranging from 0.0001 to 0.001 ng/μL, with no cross-reactivity against 18 reference species. These methods were further refined to create a multiplex ultrafast RT-PCR technique that could simultaneously identify seven shrimp species within 23 min while maintaining analytical accuracy that can be adapted for various analytical settings, field applications, rapid screening, quality control, and regulatory compliance, making them invaluable tools for the food industry and regulatory authorities.

A new species of Proaphelinoides Girault (Hymenoptera, Aphelinidae) from China, with a phylogenetic analysis.

A new species of Proaphelinoides Girault, Proaphelinoideshuangi Chen & Jiang, sp. nov., is reported from China. A key to all species of the genus is provided. DNA standard barcode COI and partial nuclear ribosomal 28S-D2 from two individuals of Proaphelinoides were sequenced, and 28S-D2 rDNA was included in a phylogenetic analysis, confirming Proaphelinoides as the sister group to Aphytis.

A DNA barcode library for woody plants in tropical and subtropical China

The application of DNA barcoding has been significantly limited by the scarcity of reliable specimens and inadequate coverage and replication across all species. The deficiency of DNA barcode reference coverage is particularly striking for highly biodiverse subtropical and tropical regions. In this study, we present a comprehensive barcode library for woody plants in tropical and subtropical China. Our dataset includes a standard barcode library comprising the four most widely used barcodes (rbcL, matK, ITS, and ITS2) for 2,520 species from 4,654 samples across 49 orders, 144 families, and 693 genera, along with 79 samples identified at the genus level. This dataset also provides a super-barcode library consisting of 1,239 samples from 1,139 species, 411 genera, 113 families, and 40 orders. This newly developed library will serve as a valuable resource for DNA barcoding research in tropical and subtropical China and bordering countries, enable more accurate species identification, and contribute to the conservation and management of tropical and subtropical forests.

DNA Barcoding Based Identification of Rosa x Damascene and Prunus dulcis Herbs Using ITS2 Barcoding Gene Amplification.

The considerable risk of adulteration in the herbs has raised commercial interest in the identification of medicinal herbs globally. DNA barcoding is the primary techniques for identifying the herbs at genetic level. This technique's key benefit is that it can identify the material's purity. This study focuses on the accurate identification of species utilizing Polymerase chain reaction-based nuclear universal internal transcribed spacer region (a barcode region) amplification and sequencing in 2 medically significant plants (Rosa damascene and Prunus dulcis) procured from the local herbal market of Karachi in the year 2022. Results suggest that the ribosomal nuclear ITS2 region of the selected plant species shows the 100 % identity with the reference genome, therefore it has shown a good rate of identification at the species level. The findings of this concludes that ITS2 is the novel standard barcode that involve in species identification, genomic conservation, and secure utilization of the medically significant plant species.

Spectral barcode based on one-dimensional photonic crystal with continuous refractive index modulation

This paper demonstrates an example of optical information encoding using the spectral response of a photonic crystal. The spectral dependence of the reflection coefficient of a one-dimensional photonic crystal reproduces the sequence of the EAN-13 standard barcode. An algorithm for building the structure of a photonic crystal based on a given encoded sequence of 12 digits was developed. This structure was experimentally implemented, its spectral response allows to decode the original encoded sequence.

Phylogenetic insight into the Lecidea atrobrunnea complex – evidence of narrow geographic endemics and the pressing need for integrative taxonomic revisions

AbstractSpecies of lichen-forming fungi (LFF) display an array of geographical distribution patterns. Among the broadly distributed lichen-forming fungal species, the degree of reproductive isolation and genetic substructure among populations varies widely, in some cases masking unrecognized diversity or meaningful biogeographical patterns. Lecidea atrobrunnea (Raymond ex Lam. & DC.) Schaer. s. lat. (Lecideaceae) is a widespread species complex that has been studied for over two centuries since its initial description. The diversity of the L. atrobrunnea group is highest in western North America, where a dizzying array of morphologies and chemistry can occur at local scales. Here we investigate whether the assumed cosmopolitan distribution of L. atrobrunnea s. lat. is an artifact of taxonomic limitations and masks biogeographical patterns in this species complex. To address these questions, we compiled sequence data from the standard fungal barcoding marker (ITS) for over 100 specimens within this complex, in addition to genome-scale data from a subset of these representing over 1600 single-copy nuclear genes spanning over 3 Mb of the genome. Our study corroborates the perspective that the morphologically and chemically variable Lecidea atrobrunnea group reflects a complex of distinct species-level lineages, with 42–83 candidate species inferred from the ITS region and high levels of diversity inferred from a subset of specimens using genome-scale data. However, both phenotype- and molecular-based species boundaries remained unsettled, with the most common nominal taxa recovered as highly polyphyletic and with conflict among different molecular species delimitation approaches. Our study also highlights the potential for geographically restricted species, with fascinating biogeographical patterns, challenging, in part, the assumed cosmopolitan distribution of L. atrobrunnea s. lat. This study provides valuable direction for future research that will be crucial in understanding diversification and establishing a robust taxonomy for this well-known species complex.

Sequencing Analysis of COI Genes Isolated from Vietnam’s Endemic Insect Species (Insecta: Plecoptera) for DNA Barcoding Database

The Insecta are the most diverse group of animals on the planet. The diversity of Plecoptera aquatic insect order is used as an environmental and ecological indicator. The study of endemic species is important for confirming the national biodiversity and contributing to the determination of national sovereignty. Despite Vietnam being a biodiversity hotspot, the number of known insect species remains limited. As a result, DNA barcoding has been used as a cutting-edge technique to efficiently and rapidly classify biological species. The Cytochrome c oxidase subunit I (COI) gene is widely regarded as a standard barcode for animal classification. In this study, we amplified and sequenced a portion of the COI gene fragment with a molecular weight of 710 bp for eight insect species belonging to the order Plecoptera, endemic to Vietnam. When compared to the previously published sequences of other Plecoptera species, the results of COI gene sequence analysis showed a high divergence of at least 9.9%. Molecular analysis of the studied species accurately demonstrated their endemism and morphological similarities. This study contributes to the development of a DNA barcoding database for endemic insects in Vietnam.

First insight into molecular diversity and DNA barcode library of epikarst-dwelling invertebrates in the Western Carpathians

DNA barcoding represents a handy tool for species identification. In addition, it serves as a complementary approach that improves the characterisation of evolutionary lineages and facilitates the detection of potentially undescribed and cryptic species. Based on the case study in the Western Carpathians, which belong to the Carpathian biodiversity hotspot, we have compiled the first DNA barcode reference library for molecular identification of invertebrates associated with epikarst, a unique, yet understudied, shallow subterranean aquatic habitat that extends at the interface between the soil and carbonate rocks. We analysed invertebrates collected in 2019-2020 from epikarst water that continuously seeps into four caves of the Demänovský Cave System in northern Slovakia. The standard barcode marker of the mitochondrial COI gene was amplified in more than 920 individuals of aquatic, semi-aquatic, and terrestrial invertebrates. The final data set consisted of 784 barcode sequences representing 36 morphospecies, the majority (98.3%) belonging to Arthropoda. Automated cluster delineation using the Barcode of Life Data System (BOLD) revealed 60 Barcode Index Numbers (BINs), of which 43 BINs were new to BOLD, representing mostly typical subterranean species. Almost 20% of the morphospecies displayed high intraspecific variation (>2.2%), suggesting the need for further investigation to assess potential taxonomic problems or cryptic diversity. Our results also indicated the existence of several yet undescribed invertebrate species and possible heteroplasmy or COI numts in the collembolan Megalothorax sp. (incertus species group). The resulting DNA barcode library represents a significant advance not only in the characterisation of epikarst biodiversity but also in the understanding of subterranean biodiversity in general, paving the way for future complex evolutionary and biogeographical studies.

A new species of minute litter bug of the genus Cryptostemma (Heteroptera: Dipsocoromorpha: Dipsocoridae) from Basse-Terre (Guadeloupe Islands)

Summary A new species of the genus Cryptostemma Herrich-Schaeffer, 1835, C. karukeraensis n. sp., was collected in several localities of the island of Basse-Terre (Guadeloupe), by prospecting the particular micro-habitats specific to the Dipsocoridae. Males and females (macropterous) and 5th instar nymphs are described and illustrated, in particular details of the pregenital abdomen, and the highly asymmetric and modified genitalia in the male. Among its American congeners, the new species shows morphological similarities with C. haywardi Wygodzinsky described from Argentina, but differs in male and female genital characters. The standard barcode DNA sequence (COI) is given for this new species, which is the third known dipsocoromorphan representative from Guadeloupe, after Ceratocombus bifenestratus Poppius, 1910 (Ceratocombidae) and Schizoptera elegans Poppius, 1910 (Schizopteridae).

Description of a new species of the genus Xyrosaris Meyrick (Lepidoptera: Yponomeutidae) from the Far East of Russia with notes on congeneric species.

A new species, Xyrosaris insularis sp. n., was found in the Far East of Russia. Most of the specimens were obtained through the rearing of larvae that fed on Celastrus orbiculatus Thunb. (Celastraceae). The genetic distances between the mtCOI sequences in X. insularis and congeneric species are in the range 1.2-13.9%. Minimal genetic distance (1.2%) was discovered between new species and X. lichneuta Meyrick from Shaanxi (China), which is lower than the standard mtCOI barcoding threshold of 2% for species delineation, but both taxa differ well in the genital morphology. The description of a new species is accompanied by illustrations of variations in the pattern, by the genitalia of both sexes, and by larva on its host plant.

Efficient multiplexed label-free detection by flexible MXene/graphene oxide fibers with enhanced charge transfer and hot spots effect

Sensors integrated with sampling and analysis processes are the key in analytical chemistry, environmental protection and national security. However, achieving reliable and sensitive large-scale rapid screening of target analytes is still challenging. Herein, we demonstrate a robust surface-enhanced Raman scattering (SERS) sensor by constructing dense and uniform plasmonic hotspots on the flexible Ti3C2Tx MXene/graphene oxide (MG) fibers through a self-assemble strategy at the oil-water interface. The produced SERS substrates exhibited an ultralow limit of detection (LOD, 1 ×10−15 M for R6G), outstanding sensitivity (EF = 1.53 ×1012) and high stability for R6G molecules (RSD = 9.47%, over 60 days storage). SERS experiments and theoretical simulations suggested that acidified Ti3C2Tx MXene further excited charge-transfer (CT) resonance of the system, while the assembled Ag nanostructures produced a large electromagnetic (EM) field enhancement. Thus, the fiber sensor not only realizes the multiplexed and powerful detection of pesticide residues (LOD of 10−11 M) with an error < 7.3% via an established concentration-dependent standard color barcodes, but also allows the recognization of target molecules in a wide range of fields, such as methylene blue, crystal violet and nikethamide. This work provides new insights in the development of versatile label-free sensors for the rapid multiplexed analysis of target molecules in real samples.