Metabarcoding and Metagenomics

Environmental DNA (eDNA) metabarcoding has become a powerful approach for large-scale biodiversity assessment, yet taxonomic assignment remains one of its most critical error-prone steps. Current bioinformatic pipelines rely on molecular similarity searches against reference databases, but assignment accuracy is constrained not only by short marker length and database incompleteness, but also by fundamental limitations, including recent species radiations, incomplete lineage sorting, introgression, NUMTs, and the imperfect correspondence between genetic variation and species boundaries. Here, we present TRIDENT (Taxonomic Resolution and IDentification using Environmental dNa Traces), an automated and simple protocol designed to improve taxonomic assignments in eDNA metabarcoding. Initially developed for marine vertebrates, TRIDENT may be used with any barcode and integrates three complementary sources of evidence: molecular similarity (NCBI/GenBank and BOLD), curated taxonomic information (WoRMS), and ecological plausibility derived from biogeographic occurrence data (GBIF). The workflow sequentially constructs candidate taxon lists based on sequence similarity, expands them through taxonomic hierarchies, and filters them using spatial occurrence constraints. It further identifies possible taxa lacking reference barcodes and evaluates their plausibility through CO1-based similarity if data exist in BOLD. TRIDENT has been implemented as a source-available Python tool and tested using empirical eDNA datasets from marine vertebrates as well as simulated communities. Results demonstrate that the tool produces taxonomic assignments consistent with expert manual curation while substantially reducing processing time and attention errors caused by manual processing of large datasets. By combining molecular, taxonomic, and ecological criteria within a single framework, TRIDENT improves transparency and reproducibility and provides a robust and flexible solution strengthening confidence in taxonomic identifications in eDNA-based biodiversity assessments.

Environmental DNA (eDNA) metabarcoding has become a cornerstone of marine biodiversity monitoring, yet it recovers genetic material irrespective of organism viability and may therefore conflate historical and contemporary community signals. Environmental RNA (eRNA), derived from less stable ribonucleic acid, is hypothesized to be biased toward metabolically active organisms and may provide a more temporally resolved snapshot of living communities. Here we present a paired eDNA/eRNA metabarcoding comparison across a tropical marine seascape, analyzing 19 co-sampled sites spanning coral reefs, mangroves, a seagrass bed, shipwrecks, a cenote, and coastal infrastructure around San Andres Island, Colombia. To our knowledge this is the first in situ, ecosystem-scale paired eDNA/eRNA survey of the broad eukaryotic community across multiple natural habitat types in a tropical marine system, extending mesocosm and freshwater work (e.g., Giroux et al., 2022) to a field setting. Using COI-region amplicon sequencing processed by NatureMetrics, we recovered 1,944 operational taxonomic units (OTUs) across the 19 paired sites. Of these, 1,015 (52.2%) were detected by both approaches, 305 (15.7%) were unique to eDNA, and 624 (32.1%) were unique to eRNA. The eRNA-unique fraction was taxonomically enriched for groups including diatoms (class Bacillariophyceae, phylum Ochrophyta), ciliates, and other protists. Paired Wilcoxon signed-rank tests showed that eRNA recovered significantly higher OTU richness (median 239 vs. 207; W = 36, p = 0.016) and Shannon diversity (median 3.64 vs. 3.38; W = 40, p = 0.026) than eDNA. The mean per-site Jaccard similarity between paired samples was 0.40, indicating substantial turnover in the rare-taxon composition recovered by each method. Principal coordinates analysis of Bray-Curtis dissimilarity showed that habitat type structured abundance-weighted community composition (PERMANOVA F = 2.49, p = 0.001) whereas molecular method did not (F = 1.37, p = 0.107). A PERMDISP test found homogeneous multivariate dispersion between methods (F = 0.01, p = 0.92), reinforcing the absence of a method effect, but significant dispersion heterogeneity among habitats (F = 24.0, p < 0.01), so the habitat result is interpreted with caution. Indicator species analysis identified 73 OTUs significantly associated with one template: eDNA indicators were dominated by dinoflagellates (Dinophyceae) and eRNA indicators by diatoms (Bacillariophyceae) and fungi, consistent with an eRNA bias toward metabolically active microbial eukaryotes. A read-weighted overlap analysis showed that although eRNA-unique OTUs outnumbered eDNA-unique OTUs roughly two to one, the large majority of reads (>95%) fell in shared OTUs, so method-unique detections are predominantly rare taxa. We discuss the complementary value of eRNA for marine monitoring, with the seagrass habitat -- where eRNA reduced masking by terrestrial plant material -- as the clearest use case, and propose, rather than prescribe, the integration of eRNA into routine programs.

The marine biodiversity inhabiting rocky shores in the Colombian Pacific remains largely undocumented, primarily due to geographic isolation, logistical challenges, and socio-political constraints. To address the existing knowledge gap, we conducted an expedition to enhance baseline biodiversity knowledge in rocky shores by integrating multiple complementary approaches, including visual censuses, specimen collection with morphological identification, environmental DNA (eDNA) metabarcoding and DNA barcodes. eDNA samples were collected at four coastal sites adjacent to rocky substrates, along with biological specimens obtained from fourteen locations through SCUBA diving at depths ranging from 1 to 25 meters. Tissue samples were subjected to genomic DNA isolation, followed by the generation and validation of cytochrome c oxidase subunit I (COI) barcode sequences, which were subsequently corroborated through taxonomic assessment to ensure accurate species identification. eDNA metabarcoding analyses yielded over 7 million high-quality sequence reads. Although taxonomic resolution at the species level was constrained by the limited completeness of reference sequence databases, a total of 106 species and 83 families were successfully identified, predominantly within the classes Actinopteri, Chondrichthyes, and marine mammals. From the 769 specimens obtained we generated 871 sequences, including 414 validated COI barcodes representing 76 species across 64 families. The integration of DNA barcoding and eDNA approaches resulted in over 1,400 taxonomic detections spanning five phyla, with only six species shared between methodologies. Richness and diversity varied among sites, and revealed significant differences along the coastline between Jurado and Cupica Gulf. All sequences were deposited in BOLDsystems database under the CCBIO project and were visualized through OBIS and GBIF databases. These findings provide the first molecular-based baseline for rocky shore biodiversity in the Colombian Pacific, highlighting the value of integrative approaches for monitoring and conservation.

Animal COI (mitochondrial cytochrome oxidase I) metabarcoding of environmental DNA (eDNA) is increasingly used to assess biodiversity in complex substrates such as soil. However, due to read-length constraints of second-generation sequencing platforms, mini-barcodes have been used instead of the full barcode region. Long-read sequencing technologies now enable the recovery of full-length barcode sequences, and are more commonly applied for studying microbes, but their use for metabarcoding the full-length standard COI barcoding region in animals remains limited. In this study, we compared three COI amplicon sets -- 313 bp, 660 bp, and 1,256 bp -- amplified from soil eDNA samples and sequenced using Illumina and PacBio platforms to evaluate their overall concurrence, the effectiveness of identifying nuclear mitochondrial DNA segments (NUMTs) and chimeras, as well as their respective taxonomic resolution. The long-read datasets exhibited a higher identification rate of NUMTs and true chimeras, suggesting that longer sequences improve the detection of noise in COI metabarcoding data, thereby reducing the occurrence of spurious taxa. Taxonomy assignment confidence was similar between the 313 bp and 660 bp datasets, whereas extending the amplicon beyond the standard COI barcode region (1,256 bp) reduced confidence, likely because longer reads extend into regions poorly represented in barcode reference databases. Despite substantially lower sequencing depth in the 660 bp dataset, per-sample OTU richness did not differ significantly from that recovered with the Illumina 313 bp amplicon set. Similarly, the relationships between samples were strongly correlated across the detected OTU communities, indicating consistent ecological interpretations between short and long amplicons. We conclude that the standard ~658 bp COI barcode is an optimal marker for soil animal metabarcoding from eDNA, balancing target recovery, artifact detection, taxonomic assignment and ecological interpretability. As COI eDNA metabarcoding becomes increasingly used in biodiversity assessment and is increasingly adopted in large-scale monitoring initiatives, this study provides methodological guidance for improving the robustness of soil animal community biomonitoring.

1. Detecting environmental DNA (eDNA) from rare or low-abundance aquatic species remains a major challenge, particularly when it is highly degraded, present at low concentrations, and dominated by DNA from non-target taxa. These challenges are further amplified in sedimentary ancient DNA (sedaDNA) studies, where thousands of years can degrade eDNA further, making the detection and quantitative interpretation of weak biological signals difficult. 2. Metabarcoding is commonly used to produce high-throughput community-level data from eDNA but is inherently compositional and influenced by amplification biases. Nonetheless, metabarcoding read abundance or PCR replicate detection frequency are increasingly used as proxies for relative DNA concentration, but their quantitative interpretation has rarely been evaluated against independent measures of absolute DNA abundance. 3. We used droplet digital PCR (ddPCR) to quantify mitochondrial DNA from Atlantic cod (Gadus morhua) and Atlantic herring (Clupea harengus) in 136 ancient eDNA extracts from Icelandic marine sediment cores spanning the last three millennia. We compared ddPCR copy number estimates with metabarcoding (18S) derived relative abundance and detection frequency, and evaluated whether temporal DNA trends corresponded with proxy reconstructed sea surface temperature (SST) variability. 4. We found that ddPCR-measured fish sedaDNA abundance was positively correlated with the proportion of metabarcoding PCR replicates for both Atlantic cod and Atlantic herring. Moreover, temporal trends in Atlantic herring DNA abundance were consistent with proxy reconstructed SST variability, supporting the ecological relevance of the molecular signal. 5. Overall, our results show that ddPCR-derived DNA concentrations and metabarcoding PCR replicate detection frequency capture consistent patterns in low-abundance fish sedaDNA from marine sediments. The observed agreement between approaches supports the use of PCR replicate detection frequency as a semi-quantitative proxy for low-abundance sedaDNA.

Here, we report the first genetic confirmation of golden mussels (Limnoperna fortunei) in North America, and the subsequent development, optimization, and deployment of golden mussel eDNA monitoring procedures. Aquatic species invasions are economically costly, disrupt ecosystem functionality, and impact native aquatic communities. Early detection of new invasive species enables rapid response via implementation of effective eradication or control measures and is key for reducing harmful outcomes. Initial species detection and taxonomic identification can be aided by genetic methods that have high detection sensitivity and accuracy. Genetic methods such as environmental DNA (eDNA) sampling can be used to detect invasive species before they become established in new systems, providing an early alert system to inform resource managers. Golden mussels were first detected in North America in October 2024 near the Port of Stockton in the San Francisco Estuary (SFE). The SFE is particularly vulnerable to invasion due to the access and connectivity provided by the presence of engineering infrastructure and shipping lanes. Collaborative efforts between public agencies and academic institutions are underway to develop a coordinated detection and response plan. Early detection followed by a rapid response is the best defense against prolific invasive species, such as the golden mussel.

Offshore industry still largely relies on traditional approaches for regulatory compliance to environmental impact on the water column. Implementing environmental DNA (eDNA) workflow can offer several advantages, but early stages such as sampling and conservation of the samples require standardization and simplification before they can be routinely applied in offshore monitoring programs. In this study, we assessed the effect of several filter types (Durapore disc, Sterivex capsule and Wattera high-capacity capsule; all with 0.22 {micro}m pore size) allowing for different volume of filtration used for sampling eDNA. We also evaluated the effect of 25 days conservation of unfiltered water samples with different preservative solutions (Benzalkonium chloride -BAC, Longmires solution LONGI and a modified Longmires solution without SDS, LNoSDS) as a viable option when immediate filtration and cold storage are not possible. For downstream eDNA evaluation of filter types and preservation, we used quantitative digital PCR on selected target DNA and metabarcoding for qualitative assessment of marine prokaryotic and eukaryotic communities. Overall, filter choice had relatively less effects on quantitative and qualitative information from eDNA compared with water preservation. Sterivex and Durapore were better filter choices for biodiversity assessment. While the Wattera filter allowed processing of larger water volumes and improved quantification of metazoan DNA, handling and processing were more challenging. For water conservation, LNoSDS was the best option. Chemical agents of LONGI and BAC may provide favourable substrates for some tolerant bacterial strains, altering the microbial community composition, with consequences for the overall qualitative evaluation of conserved eDNA. For targeted metazoan eDNA, however, chemical preservation showed clear benefits. This research highlights key considerations and viable options for eDNA sampling and simple preservation workflows without cold storage for implementation in offshore water column monitoring. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733101v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@22a175org.highwire.dtl.DTLVardef@1960864org.highwire.dtl.DTLVardef@1010f49org.highwire.dtl.DTLVardef@92a2f6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINeed for standardization of eDNA workflow for offshore water column monitoring C_LIO_LIImportance of eDNA sampling (filters) and eDNA conservation (preservatives) C_LIO_LIFilter choice does not affect drastically the dominant eDNA communities C_LIO_LIConservation outside cold storage challenging for eDNA-based biodiversity evaluation C_LIO_LIViable options: Sterivex filter for sampling; Longmires (no SDS) for conservation C_LI

DNA barcoding involves the recovery of a DNA sequence for a target gene region from its source specimen. This process gains complexity when multiple sequences are recovered from a specimen, as is often the case when data are generated by high-throughput sequencers. This diversity can reflect both methodological artifacts (e.g., chimeras, PCR errors, sequencing errors, tag jumps) and real template diversity in the DNA extract (e.g., contamination, endosymbionts, NUMTs, parasites). To support analysis of the sequence data from three million specimens annually, the Centre for Biodiversity Genomics (CBG) has developed BIP, the Barcode Inference Pipeline. Compatible with all sequencing platforms, BIP processes .fastq files and returns both target DNA barcodes and non-target sequences. To generate results, BIP implements quality and size filtration, demultiplexing, primer trimming, chimera scanning, sequence error correction, OTU delineation, and sequence identification. When analysis targets the cytochrome c oxidase 1 (COI) barcode region, BIP also assigns each OTU to a known BIN or identifies its nearest neighbour BIN. As final output, BIP returns summary files ready for upload to BOLD or for other downstream analyses. They include a taxonomic assignment for each OTU, generated by comparison with a DNA barcode reference library. We describe BIPs flexibility and structure, then demonstrate its functionality by analyzing COI sequence data from 100K specimens. Because of its capacity to disentangle target and non-target sequences, BIP outperforms an alternative software package, ONTbarcoder, in several important ways. To ease access, installation, and functionality, BIP is provided as a Docker container (github.com/cbg-innov/BIP).

Environmental DNA (eDNA) methodology is widely applied in the biomonitoring of organisms, but it requires the target DNA to be detected in a simple, stable, and highly sensitive manner. Detection sensitivity of eDNA measurement becomes particularly critical when monitoring species present at low abundance. In this study, we aimed to improve the detection sensitivity through a method of DNA-extract reconcentration. This approach involves reconcentrating eDNA samples that were originally extracted using the widely adopted DNeasy Blood and Tissue Kit (Qiagen), utilizing the same kits reagents, and does not require any additional equipment or reagents. We evaluated the ability of this DNA reconcentration method using field samples including river, lake and costal marine habitats. Evaluation of this DNA reconcentration method showed that when ten conventionally extracted samples were pooled, the DNA concentration increased by approximately sevenfold, as confirmed by DNA quantification and quantitative PCR analyses, demonstrating enhanced detection sensitivity.

Passive sampling is the deployment of a collection material in the environment to continuously capture environmental DNA (eDNA) over time, offering the potential to integrate biodiversity signals while reducing the need for repeated active water collection. However, the mechanisms governing eDNA capture and retention on passive samplers remain poorly understood, limiting the interpretation of passive eDNA signals and their broader application. Here, we investigated the mechanistic performance of glass fibre passive samplers using controlled mesocosm experiments with three invasive freshwater bivalves: zebra mussels (Dreissena polymorpha), quagga mussels (Dreissena bugensis), and Asian clams (Corbicula fluminea). Specifically, we quantified eDNA accumulation dynamics, evaluated the contribution of different eDNA states, tested the persistence of captured eDNA, and compared passive sampler signals with conventional active sampling. Passive samplers rapidly accumulated target eDNA within hours of deployment, after which concentrations either plateaued or continued to increase depending on species. Sequential transfer of passive samplers between mesocosms containing different species showed that previously captured eDNA declined while new target eDNA accumulated to concentrations comparable to freshly deployed samplers, demonstrating continual turnover rather than permanent retention. Dissolved eDNA showed little evidence of accumulation beyond the concentration retained in the pore water within the membrane, suggesting that it is unlikely to be the dominant contributor to long-term passive sampler signals. Instead, the observed variability among replicate samplers, together with the physical properties of glass fibre membranes, suggests that membrane-bound and particulate eDNA are the primary contributors to passive eDNA capture. Collectively, these findings support a model in which glass fibre passive sampler signals reflect a dynamic equilibrium between ongoing eDNA capture and concurrent loss processes rather than cumulative accumulation over time. This mechanistic framework provides a foundation for interpreting passive eDNA data and informs the future development of passive sampling materials, deployment strategies, and biodiversity monitoring applications.

Among eukaryotes, insects are by far the most diverse organisms on Earth, yet their global decline threatens ecosystem stability. Understanding local and regional biodiversity patterns is critical for conservation planning, ecosystem management, and predicting responses to environmental change, but traditional surveys for assessing insect diversity (e.g., manual collection, morphological identification, and counting) are highly labor-intensive, time-consuming, and often require rare or simply unavailable dedicated taxonomic expertise. DNA metabarcoding offers an efficient, high-resolution alternative to assess insect communities. Here, we report on the first insect metabarcoding survey on Crete that spans two years of sample collection between 2021 and 2023 from a small area in Southern Central Crete in the context of a citizen science project. A total of 29 samples yielded 10,865 Exact Sequence Variants (ESVs), 10,516 of which were assigned to insects, covering 988 species, 900 genera, and 227 families across 14 orders. A comparison with the existing observation records reveals 406 potential newly-observed species and an estimated 690 unclassified species, indicating substantial cryptic diversity. Our results demonstrate that even small-scale sampling can unravel substantial insect diversity and highlight critical gaps in barcode reference databases. Our study demonstrates how DNA metabarcoding can accelerate biodiversity discovery and monitoring in understudied regions.

Current metabarcoding pipelines are inflexible with respect to study design and are poorly suited to long-read sequence data. To address these limitations, we developed MAP, the Metabarcoding Analysis Pipeline, which is a sequence-to-answer workflow supporting the analysis of amplicons from highly multiplexed and replicated study designs. Although MAP can analyze amplicons of any length from any genetic marker, it includes several features tailored to long-read COI metabarcoding. MAP installs from a Docker container and requires only sequence data, a parameters file, and a reference library. It produces intuitive reports, enabling users to evaluate their data immediately after analysis. We validate MAP by showing that it generates biodiversity estimates that correspond closely to a ground-truth dataset of single-specimen DNA barcode data and by demonstrating that it outperforms alternative platforms for COI metabarcoding. MAP is free, open-source, and available from: https://github.com/cbg-innov/MAP.

DNA metabarcoding of faeces is a powerful, non-invasive method for assessing predator diets. However, when studying the diet of generalist predators, broad PCR primers are used to amplify the wide range of potential prey species and metabarcoding outputs are often dominated by sequences from the predator. While blocking primers can be used to reduce PCR amplification of predator DNA, they frequently cause partial predator suppression and unintended prey blocking. Peptide nucleic acid (PNA) clamps, offer a promising, underutilised alternative by binding strongly and selectively to predator DNA to block its PCR amplification. In this study we designed and validated a novel PNA clamp targeting the 18S rRNA gene to suppress bird and mammal predator DNA in dietary samples. We tested this clamp on tissue mixtures and faecal samples from three seabird and two seal species across temperate, subantarctic, and Antarctic regions. The PNA clamp substantially increased the proportion of prey reads recovered while maintaining consistent prey community composition across all predator species. Our results demonstrate not only the general effectiveness of PNA clamps over standard blocking primers, but also provide a powerful, broadly applicable new tool to improve the accuracy in DNA diet metabarcoding studies.

Chinese Bahaba (Bahaba taipingensis, Sciaenidae) is a Critically Endangered, Grade I state-protected marine fish in China. Reliable, non-invasive monitoring tools are urgently needed to inform conservation and restocking efforts. We developed and validated three TaqMan probe-based qPCR assays targeting mitochondrial 12S rRNA, ND5, and control region (D-loop) loci for species-specific detection of B. taipingensis. Primer-probe sets were designed from complete mitochondrial genomes and evaluated in silico against GenBank and in vitro against tissue DNA from B. taipingensis and closely related sciaenids, plus positive eDNA samples; sensitivity was quantified using serial dilutions of synthetic target DNA. All three assays showed high specificity and consistent amplification; 12S rRNA assay (Bhb_12S) was selected for field screening based on superior low-copy sensitivity and minimal by-products. We applied the Bhb_12S assay to 414 seawater samples collected from 23 sites in western Hong Kong during 2025 surveys, detecting B. taipingensis eDNA in the September samples (0.71-2.85 copies per L; mean 2.05), with positive detections confirmed by Sanger sequencing. Overall, these validated qPCR assays provide a robust molecular toolkit for non-invasive monitoring of B. taipingensis and will aid conservation planning, restocking evaluation, and long-term biodiversity assessments in the Pearl River Estuary and adjacent coastal waters.

Lake Constance is a pre-Alpine, monomictic, oligotrophic lake situated at the southern end of Germany composed of two main water bodies: deep, oligotrophic Upper Lake Constance (ULC) and the shallow, more mesotrophic Lower Lake Constance (LLC). To date, no sequencing-based study exists of the seasonal succession of the microbial plankton in Lake Constance. Over one-year, microbial plankton communities were sampled biweekly from the top 20 m of the water column in both sites and separated into nanoplankton (NP) and picoplankton (PP). Communities were analysed using rDNA amplicon sequencing: NP samples were analysed by 18S rDNA, and PP samples by 18S and 16S rDNA sequencing. Temporal community diversity was compared between sites and the effect of two major environmental perturbations, winter vertical mixing in ULC and oxygen depletion of the bottom-water layer in LLC, on the community was examined. Despite strong environmental contrasts, microbial plankton communities exhibited conserved seasonal temporal dynamics across basins. In contrast, pronounced compositional shifts occurred during mixing and oxygen depletion events. Approximately 20% of detected taxa were positively associated with these events, with log fold changes reaching 9.82, reflecting rare or undetectable taxa outside these periods. Taxa favoured by these perturbations commonly exhibited high metabolic flexibility, including mixotrophy, fermentation, or anaerobic respiration, or possessed functional traits conferring tolerance to altered redox and mixing regimes. Our results suggest that the temporal dynamics of freshwater microbial plankton communities are driven by deterministic processes and highlight the profound impact of large, and less known, environmental changes on these communities.

Health monitoring is essential to ensure laboratory animal welfare and the reliability of experimental data in zebrafish research facilities. Conventional surveillance strategies based on resident or sentinel fish have limitations in terms of sensitivity and animal use, highlighting the need for alternative approaches consistent with the 3Rs principles. In this perspective, the present study evaluated the use of sludge collected from recirculating aquaculture systems as an environmental matrix for molecular health monitoring. Because sludge accumulates microorganisms and organic material from the entire system, it represents a promising sample for pathogen detection. Following an initial environmental surveillance phase to detect pathogens present in the system, the study aimed to optimise a molecular protocol for sludge analysis. To this end, four commercial DNA extraction kits were evaluated to assess their effectiveness in recovering bacterial DNA from sludge. Their performance was analysed by quantitative PCR in terms of extraction efficiency, repeatability, and limit of detection. The results highlighted the strengths and limitations of each DNA extraction protocol and confirmed the suitability of sludge as a non-lethal matrix for pathogen detection. These findings support the implementation of environmental monitoring as a practical and cost-effective alternative to sentinel-based surveillance, improving pathogen detection while reducing animal use in accordance with the 3Rs principles and Directive 2010/63/EU.

Octocoral colonies with unusual morphology were detected in September 2022 and October 2023 in two coastal areas east of Havana, Cuba, and tentatively identified as Unomia stolonifera. U. stolonifera is an invasive octocoral from the Indo-Pacific that was first reported in the Caribbean off Venezuela in the 2000s, where it has spread rapidly, smothering coral reefs and substantially altering benthic communities. After obtaining tissue samples from a Cuban octocoral colony, we re-examined the specimen using molecular barcoding of three mitochondrial regions (16S/ND2, mtMutS, COI) and the nuclear large ribosomal subunit (28S rRNA), and we unequivocally identified it as Xenia umbellata. X. umbellata, a native of the Red Sea, was first identified in southern Puerto Rico in October 2023 and has since been found in various marine ecosystems along the southern coast of the island. The presence of either invasive octocoral species in Cuba or elsewhere in the Caribbean would be of a serious environmental concern due to their documented tolerance, totipotentiality, propagation capacity and significant negative interactions with local benthic fauna. Attempts to eradicate the invasive soft coral colonies from Cuban waters have been initiated with apparent success, helping to control further expansion. The most likely introduction pathway is the accidental or intentional releases from the aquarium trade but transport via ballast water cannot be ruled out. We cannot discount the possibility of independent invasion events from different routes to Puerto Rico and Cuba occurring within a year of each other. Propagation from Cuba to Puerto Rico, or vice versa, which we consider highly improbable, would likely imply that soft coral populations may also have been established on Hispaniola but have remained undetected in the Dominican Republic and Haiti to date.

Microbiome analysis has become a pivotal tool in understanding the role of microbiota in human health and disease. However, the lack of standardized workflows, together with the limitations of proprietary software solutions, hampers reproducibility and flexibility. Here, we present Mbiome, an open-source, user-friendly and automated workflow designed to streamline amplicon-based microbiome analysis. Built upon QIIME2, Mbiome supports both bacterial (16S rRNA) and fungal (ITS) profiling, and is compatible with raw fastq files generated by Ion Torrent (IT) and Illumina (IL) sequencing platforms. The workflow guides users through an interactive setup process via a simple configuration file, enabling researchers with minimal bioinformatics experience to perform comprehensive analyses without writing code. Once configured, Mbiome automates major steps including quality control, taxonomic assignment, and {beta}-diversity analyses, functional predictions (via q2-metnet), and customizable visualizations and statistical analyses. Mbiome has been validated using real-world datasets from multiple sclerosis research projects, performing a comparison between different microbiome analysis approaches, including 16S hypervariable region reconstruction, amplicon-based strategies, and cross-platform sequencing (IT and IL), as well as against results obtained with Ion Reporter (IR) commercial software. This evaluation demonstrated its versatility and effectiveness across different sequencing platforms. Moreover, Mbiome provided improved flexibility, transparency, and taxonomic resolution compared to IR. By combining accessibility, reproducibility, and cross-platform compatibility, Mbiome lowers the barrier to microbiome data analysis and facilitates high-quality, standardized workflows in both research and applied settings. Mbiome is publicly available at https://github.com/MGorostidi/mbiome.

1. Most species are neither discovered nor named, let alone included in analyses that require biological information such as trait measurements, images, ecological information and genome-scale data. Specimen-level DNA barcoding can help discover many of these species rapidly, but everything beyond discovery requires vouchers organized into putative species. Yet, existing barcoding workflows lack efficient techniques for voucher recovery, creating a post-barcoding bottleneck that limits the ability of converting barcoded specimens into biological knowledge. 2. Here we present a low-cost, open-source workflow consisting of two stages. The first safeguards barcoded specimens by separating them from DNA extracts and transferring them from microplates into ethanol-filled glass vials. The second converts the resulting voucher collection into a searchable physical resource by linking barcode-derived molecular Operational Taxonomic Unit (mOTU) assignments to vial positions and enabling specimens to be sorted into putative species either manually or automatically using a newly developed open-access robot (SORTER). 3. We evaluated the workflow using 2,024 insect specimens distributed across 21 96-well plates. For the first stage, DNA separation and specimen transfer required approximately 15 minutes per plate. For the second stage, MOTUmapper generated retrieval coordinates in a few seconds, after which the 2,024 vouchers belonging to the 452 putative species could be recovered manually in 5 days or with SORTER in 5 hours. Throughout both stages, specimen identities remained linked to barcode sequences, metadata and storage positions. 4. Vouchers are the Rosetta stones of biology because they connect different kinds of data to the same specimens. By safeguarding these vouchers and making them searchable, the workflow converts barcode projects from one-time molecular surveys into reusable resources for ecological and evolutionary research.

Ranges of small benthic fauna are notoriously difficult to assess. In some of these cases, modern eDNA methods can shed light on species occurrence. Here we conduct an exploratory study on the fish eDNA recoverable from the gastrovascular cavities of the easy-to-sample pore water siphoning benthic invertebrate, Cassiopea, across six sites within the Florida Keys. Twenty-seven fish 12S identities were recovered from water samples, two from sediment samples, and seventeen from Cassiopea gut swabs. In total, thirty-two different species were identified from nineteen families, including one shark species (Ginglymostoma cirratum), and five species of cryptobenthic reef fishes (f: Gobiidae, Labrisomidae). Additionally, five species were identified from medusae samples that were not recovered in water or sediment samples. The species identities recovered may provide insight into the fish in direct proximity to Cassiopea assemblages, as well as indicate that Cassiopea may accrue disproportionate eDNA from cryptobenthic reef fish compared to surrounding environmental samples. The unorthodox sampling technique of using eDNA recovered from jellyfish stomachs yields another avenue for epibenthic community data acquisition.