DNA Barcoding Survey Research Articles

First DNA Barcoding Survey in Bulgaria Unveiled Huge Diversity of Yeasts in Insects.

In this study, we conducted a comprehensive survey aimed at assessing the diversity of yeast species inhabiting the guts of various insect species collected mainly from two Bulgarian National Parks, namely, Rila, and Pirin. The insect specimens encompass a broad taxonomic spectrum, including representatives from Coleoptera, Orthoptera, Lepidoptera, Hymenoptera, Dermaptera, Isopoda, and Collembola. Yeast strains were identified with DNA barcoding using the ribosomal markers, specifically, the D1/D2 domains of the ribosomal large subunit (LSU) and the internal transcribed spacers regions ITS 1 + 2 (ITS). The analysis unveiled the presence of 89 ascomycetous and 18 basidiomycetous yeast isolates associated with the insect specimens. Furthermore, our study identified 18 hitherto unknown yeast species.

DNA Barcoding Revealed a High Percentage of Mislabeling in Commercial Fish Products: The First Empirical Survey in South Texas

Seafood mislabeling is a widespread problem that have produced a growing distrust of seafood industry. In this study, we examined the prevalence of mislabeling in fish samples from regional markets in the greater Houston area and close-by coastal communities. A total of 63 fish fillet samples were purchased, labeled, and stored at -20˚C in individual packages until DNA extraction. DNA fragments of roughly 700 base pairs encoding cytochrome oxidase I (COI) were PCR-amplified from each DNA sample. With 99.6–100% nucleotide identity in the GenBank and BOLD databases, all samples were correctly identified at the species level. The scientific names identified by DNA barcoding were matched with legally acceptable market names using the US Food and Drug Administration (FDA) Seafood List. Out of the 63 samples examined, 13 samples (20.6%) were mislabeled. Moreover, 24 samples (38.1%) did not use the acceptable market names, indicating that the FDA policy is poorly implemented in seafood industry. The first DNA barcoding survey this area warranted the need of continuous monitoring and the dissemination of the regulation combined with taxonomic knowledge.

The State of Knowledge of CCA Diversity in the Caribbean Coral Reefs

Crustose coralline algae (CCA) are a diverse and ecologically important species found in most of the world’s oceans. The current lack of taxonomic knowledge and relative abundance compromises our ability to predict species diversity numbers and, thus, their ecological roles and impacts on coral reefs. To gather a better understanding of the state of knowledge of crustose coralline algae taxonomy in the Caribbean, 107 different research papers, and other primary and secondary literature were studied; any source with taxonomical information, species identification, or genetic markers for identification was recorded. All Genebank codes were collected and sorted by supposed species marker and then ran through the National Center of Biotechnology Information. The location these genetic markers were gathered from was compared to the natural habitat range of the species, based on the Algaebank habitat description. Of the supposed 83 described species of crustose algae in the Caribbean, based on morphological characteristics, only 24 total were confirmed by DNA markers. This leaves at least 59 species of CCA to be confirmed in the Caribbean Sea with molecular markers. This indicates the importance of DNA barcode survey studies to assess the accurate diversity of this group in the region. With this limited knowledge apparent, it should be seen that a CCAs phylogenetic and taxonomical review must be done. An in-depth assessment should be conducted on CCA collections to identify the Caribbean species correctly and thus know their biodiversity in local habitats.

Limited phylogeographic structure in a flightless, Appalachian chalcidoid wasp, Dipara trilineata (Yoshimoto) (Hymenoptera, Diparidae), with reassessment of the male of the species

Dipara trilineata (Diparidae) is a widespread eastern North American parasitoid with apterous females and winged males. Despite its seemingly limited dispersal capabilities, phylogeographic analysis over southern Appalachia reveals little structure, with only limited population level isolation. DNA barcoding surveys also definitively associate the male of the species, which had previously been misattributed, and a description of the correctly associated male is provided.

DNA barcoding of marine teleost fishes (Teleostei) in Cebu, the Philippines, a biodiversity hotspot of the coral triangle

A morphology-based barcoding library of market teleost fishes (Teleostei) in Cebu is built based on cytochrome c oxidase subunit I (COI) sequences and voucher specimens which aimed to establish a reliable reference of frequently traded fishes in the province, a biodiversity hotspot at the center of the Philippine archipelago. A total of 1721 specimens were collected from 18 fish markets and landing sites around the province, in which 538 specimens were sequenced belonging to 393 species from 229 genera, 86 families, and 37 orders. Most speciose families are coral reef or reef-related shallow-water species. Twelve species from 11 families are newly recorded in the Philippine waters, among which 7 species are deep-sea inhabitants, while 3 species have expanded their distribution range. Only 20 taxa could not be identified to the species level due to the difficulty in morphological examinations, absence of matched reference sequences in online databases, and/or problematic species awaiting further studies. This first comprehensive DNA barcoding survey of Cebu fishes can facilitate further taxonomic research as well as the conservation and management of fisheries in the Philippines.

Morphological and molecular investigations shed light on diversity and distribution of Palmariaceae in the north-western Pacific

ABSTRACT Traditional morphology-based investigations of the red algal genera Devaleraea and Palmaria (Palmariaceae, Rhodophyta) fail to provide accurate accounts of diversity and distribution for species from the Russian coast of the north-western Pacific. Consequently, there is much disagreement and taxonomic uncertainty regarding several species. To resolve these issues we conducted a molecular-assisted investigation that included a DNA barcode survey (COI-5’) and multilocus (COI-5’, cob, psaA, ITS) phylogenetic analyses. Our analyses indicate that diversity was previously underestimated for the Devaleraea/Palmaria complex. We describe three additional species of Devaleraea: D. sakhalinensis sp. nov., D. kussakinii sp. nov. and D. urupiana sp. nov. We consider D. titlyanoviorum to be an alga with abundant proliferations from the margins, branching to several orders, that is known only from Kunashir Island. Algae from Sakhalin Island and the mainland coast of the Sea of Japan previously identified as D. titlyanoviorum are described here as D. sakhalinensis. We confirmed that the genus Palmaria is not monotypic. It includes at least two species: P. palmata in the North Atlantic and P. moniliformis in the north-western Pacific. We expand the known ranges of D. callophylloides, D. mollis and P. hecatensis in the north-western Pacific southwards and find that D. stenogona is confined to the northern part of the Sea of Japan. The distribution patterns of species of the genera Devaleraea and Palmaria are discussed in relation to the oceanography of the north-western Pacific. HIGHLIGHTS Three new species of the genus Devaleraea are proposed. The genus Palmaria is not monotypic and includes at least two species, P. moniliformis and P. palmata.

A DNA barcode survey of insect biodiversity in Pakistan.

Although Pakistan has rich biodiversity, many groups are poorly known, particularly insects. To address this gap, we employed DNA barcoding to survey its insect diversity. Specimens obtained through diverse collecting methods at 1,858 sites across Pakistan from 2010–2019 were examined for sequence variation in the 658 bp barcode region of the cytochrome c oxidase 1 (COI) gene. Sequences from nearly 49,000 specimens were assigned to 6,590 Barcode Index Numbers (BINs), a proxy for species, and most (88%) also possessed a representative image on the Barcode of Life Data System (BOLD). By coupling morphological inspections with barcode matches on BOLD, every BIN was assigned to an order (19) and most (99.8%) were placed to a family (362). However, just 40% of the BINs were assigned to a genus (1,375) and 21% to a species (1,364). Five orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera) accounted for 92% of the specimens and BINs. More than half of the BINs (59%) are so far only known from Pakistan, but others have also been reported from Bangladesh (13%), India (12%), and China (8%). Representing the first DNA barcode survey of the insect fauna in any South Asian country, this study provides the foundation for a complete inventory of the insect fauna in Pakistan while also contributing to the global DNA barcode reference library.

DNA barcoding provides new insights on the distribution, systematics and conservation of the freshwater genus Pelasgus (Cypriniformes: Cyprinidae) in Greece

The freshwater ichthyofauna of Greece is characterized by high diversity and endemism and faces various conservation challenges. Despite significant efforts, mainly with traditional assessment methods, the precise taxonomic status and geographic range of some of the currently acknowledged fish species remain insufficiently explored. Here, the results of the first comprehensive DNA barcoding survey of the genus Pelasgus are presented, covering its entire known range in Greece. Two hundred and forty COI sequences (retrieved from collected samples, museum specimens and the GenBank) were analysed to determine the number of putative species and delineate their current distribution. Data support the existence of seven haplogroups, corresponding to the currently valid Pelasgus species. Analysis of museum samples of the critically endangered species P. epiroticus from its type locality (now extinct there) uncovers its presence in contemporary samples from a neighbouring aquatic system (Zaravina Lake) and from aquatic systems well outside its native range (possibly as a result of past translocations) for the first time. A subgroup of P. epiroticus haplotypes was also identified in North-western Greece in regions previously believed to host P. thesproticus or P. stymphalicus. Based on the above, hotspots for further Pelasgus taxonomic investigations are identified and conservation actions are discussed.

A DNA Barcoding Survey of an Arctic Arthropod Community: Implications for Future Monitoring.

Accurate and cost-effective methods for tracking changes in arthropod communities are needed to develop integrative environmental monitoring programs in the Arctic. To date, even baseline data on their species composition at established ecological monitoring sites are severely lacking. We present the results of a pilot assessment of non-marine arthropod diversity in a middle arctic tundra area near Ikaluktutiak (Cambridge Bay), Victoria Island, Nunavut, undertaken in 2018 using DNA barcodes. A total of 1264 Barcode Index Number (BIN) clusters, used as a proxy for species, were recorded. The efficacy of widely used sampling methods was assessed. Yellow pan traps captured 62% of the entire BIN diversity at the study sites. When complemented with soil and leaf litter sifting, the coverage rose up to 74.6%. Combining community-based data collection with high-throughput DNA barcoding has the potential to overcome many of the logistic, financial, and taxonomic obstacles for large-scale monitoring of the Arctic arthropod fauna.

Phylogeographic evidence that the distribution of cryptic euryhaline species in the Gambusia punctata species group in Cuba was shaped by the archipelago geological history

The main drivers of diversification of freshwater fishes in Cuba are not yet well understood. For example, salt tolerance was thought as the main factor involved in the diversification of Gambusia punctata species group in this archipelago. However, evidence from a recent DNA barcoding survey suggested the presence of cryptic species and no correlation between species delimitation and level of salinity. In this study, we analyzed the cryptic diversification of G. punctata species group in Cuba, based on a comprehensive sampling of its distribution and including habitats with different salinity levels. We evaluated the patterns of molecular divergence of the samples by sequencing a set of mitochondrial DNA (mtDNA) regions and genotyping nine nuclear microsatellite loci. We also used cytochrome b gene (cytb) partial sequences and these microsatellite loci to analyze population structure inside putative species. Five mtDNA well-differentiated haplogroups were found, four of them also identified by the analysis of the microsatellite polymorphism which corresponds to two already recognized species, G. punctata, and G. rhizophorae, and three putative new species. The extent of hybrid zones between these groups is also described. In each group, populations inhabiting environments with contrasting salinity levels were identified, indicating a generalized trait not specific to G. rhizophorae. The geographic distribution of the groups suggested a strong association with major relict territories of the Cuban Archipelago that was periodically joined or split-up by changes in seawater levels and land uplifts. Salinity tolerance might have facilitated sporadic and long-distance oversea dispersal but did not prevent speciation in the Cuban archipelago.

A DNA barcode survey of marine macroalgae from Bergen (Norway)

ABSTRACTSafeguarding the services provided by marine forests inherently depends on an accurate understanding of macroalgal species diversity. Here, we provide the first DNA barcode survey of marine macroalgae from Norway, with a focus on the Bergen area, and compared our findings to morphological listings for the corresponding location. Specimens were sampled 14–20 April and 3–13 June 2016, and variously sequenced for several genetic markers, including the five prime end of the cytochrome c oxidase subunit I gene (COI-5P), elongation factor tufA, and the ribulose-1, 5-biphosphate carboxylase large subunit gene (rbcL). We generated 655 new barcode records for COI-5P, 11 for tufA, and 50 for rbcL, representing nine species of Chlorophyta, 51 species of Phaeophyceae, and 74 species of Rhodophyta. Sequence data confirmed the presence of 113 morphological species. A further 17 genetic groups indicated the presence of new species for the area, only six of which were linked to formally described species. The remaining four genetic records were uncertain in terms of morphological species assignment and relation to the local flora. We recommend further DNA barcoding surveys in the area, as only a third of the listed morphological species were genetically confirmed.

Dark Ophiuroid Biodiversity in a Prospective Abyssal Mine Field

The seafloor contains valuable mineral resources, including polymetallic (or manganese) nodules that form on offshore abyssal plains. The largest and most commercially attractive deposits are located in the Clarion Clipperton Fracture Zone (CCZ), in the eastern Pacific Ocean (EP) between Hawaii and Mexico, where testing of a mineral collection system is set to start soon [1]. The requirement to establish pre-mining environmental management plans has prompted numerous recent biodiversity and DNA barcoding surveys across these remote regions. Here we map DNA sequences from sampled ophiuroids (brittle stars, including post-larvae) of the CCZ and Peru Basin onto a substantial tree of life to show unprecedented levels of abyssal ophiuroid phylogenetic diversity including at least three ancient (>70 Ma), previously unknown clades. While substantial dark (unobserved) biodiversity has been reported from various microbial meta-barcoding projects [2,3], our data show that we have considerably under-estimated the biodiversity of even the most conspicuous mega-faunal invertebrates [4] of the EP abyssal plain.

Description of two new Caribbean species from the Hypnea musciformis complex (Cystocloniaceae, Rhodophyta)

Hypnea musciformis is a red macroalgal species with economic importance as a source for production of carrageenan. Recent phylogeographic studies revealed a hidden diversity of cryptic species, putting in check its cosmopolitan distribution. The DNA barcode marker COI-5P and plastid rbcL, in addition to morphological studies, were used to investigate the species in this complex and to compare them with specimens from other countries including the type locality in Italy. The divergences between sequences within the H. musciformis complex were significantly high for both markers. Based on these analyses two new species are described, Hypnea caraibica Nauer, Cassano & M.C. Oliveira sp. nov. and Hypnea schneideri Nauer, Cassano & M.C. Oliveira sp. nov. Hypnea caraibica seems to be a common, widespread species in the Gulf of Mexico and the Caribbean and was recently introduced into Hawaii. This species was unnoticed due to lack of phylogeographic studies in the region. Hypnea schneideri seems to be more geographically restricted, but further surveys are needed to understand the distribution of this species. Because the identification of Hypnea species is complicated by their relatively simple and plastic morphology, DNA barcoding surveys and other molecular studies are essential to uncover hidden biodiversity in the genus and to supplement traditional studies based on morphology.

A molecular survey of Ralfsia sensu stricto (Ralfsiales, Phaeophyceae) in Canada uncovers three new species: R. robertii sp. nov., R. tenebris sp. nov., and R. unimaculata sp. nov.

Currently there are three species (i.e., traditional morphospecies) in Canada assigned to the genus Ralfsia sensu lato, which we establish is not monophyletic even after excluding “species” now known to apply to alternate stages in the life histories of non-ralfsialean species. These species include R. fungiformis (Gunnerus) Setchell & N.L.Gardner and R. pacifica Hollenberg in the Pacific, and R. fungiformis and R. verrucosa (J.E.Areschoug) J.E.Areschoug in the Atlantic. However, a DNA barcode survey of Ralfsiales in Canada using the markers COI-5P and rbcL-3P revealed five genetic groups assignable to Ralfsia sensu stricto (i.e., includes only species assigned to this genus based on phylogenetic analyses). Further complicating matters, of the three species listed previously as occurring in Canada only the type species R. fungiformis is assignable to Ralfsia sensu stricto. Ralfsia pacifica and R. verrucosa did not group with the generitype and thus are not assignable to Ralfsia sensu stricto; further, they did not group with the family Ralfsiaceae. They will be presented in a subsequent manuscript. The four other genetic groups in Canada assignable to Ralfsia sensu stricto are new species of which three are described here: Ralfsia robertii sp. nov., Ralfsia tenebris sp. nov., and Ralfsia unimaculata sp. nov. The fourth consisted of a single sterile specimen from Haida Gwaii, British Columbia, and was not characterized.

A COI DNA barcoding survey of Pratylenchus species in the Great Plains Region of North America

Abstract Pratylenchus species are among the most common plant parasitic nematodes in the Great Plains Region of North America. Our goal was to survey Pratylenchus species diversity across the Great Plains region using a mitochondrial COI DNA barcode. The objectives were to (i) determine species boundaries of the common Pratylenchus species within the region, (ii) assess the host associations of the barcoded Pratylenchus specimens, and (iii) determine Pratylenchus distribution patterns throughout the region. A total of 860 soil samples, primarily associated with eight major crops, were collected from Colorado, Kansas, Montana, Nebraska, North Dakota, and Wyoming. From this total, 246 soil samples provided the majority of 915 individual nematode specimens that were amplified by PCR and sequenced for a 727 to 739 bp region of COI. Maximum likelihood, neighbor-joining, and Bayesian phylogenetic trees all recognized 19 distinct and well-supported haplotype groups. The most common and widespread haplotype group, representing 53% of all specimens was P. neglectus, detected from 178 fields in 100 counties and associated with fields growing wheat, corn, dry beans, barley, alfalfa, sugar beets, potatoes, and a vineyard. The second most prevalent haplotype group was P. scribneri, representing 30% of all specimens and recovered from 104 fields in 45 counties, and most frequently associated with corn. Mixed field populations were encountered in approximately 20% of infested fields, with P. neglectus and P. scribneri most often occurring together in corn-soybean cropping systems. Less frequently encountered from the region were P. hexincisus, P. thornei, P. penetrans, P. alleni, and P. zeae. Eight additional haplotype groups, potentially new Pratylenchus species, were discovered in the survey.

DNA barcoding a taxonomically complex hemiparasitic genus reveals deep divergence between ploidy levels but lack of species-level resolution

DNA barcoding is emerging as a useful tool not only for species identification but also for studying evolutionary and ecological processes. Although plant DNA barcodes do not always provide species-level resolution, the generation of large DNA barcode data sets can provide insights into the mechanisms underlying the generation of species diversity. Here, we study evolutionary processes in taxonomically complex British Euphrasia (Orobanchaceae), a group with multiple ploidy levels, frequent self-fertilization, young species divergence and widespread hybridization. We use a phylogenetic approach to investigate the colonization history of British Euphrasia, followed by a DNA barcoding survey and population genetic analyses to reveal the causes of shared sequence variation. Phylogenetic analysis shows Euphrasia have colonized Britain from mainland Europe on multiple occasions. DNA barcoding reveals that no British Euphrasia species has a consistent diagnostic sequence profile, and instead, plastid haplotypes are either widespread across species, or are population specific. The partitioning of nuclear genetic variation suggests differences in ploidy act as a barrier to gene exchange, while the divergence between diploid and tetraploid ITS sequences supports the polyploids being allotetraploid in origin. Overall, these results show that even when lacking species-level resolution, analyses of DNA barcoding data can reveal evolutionary patterns in taxonomically complex genera.

Initiating DNA barcoding of Eastern Mediterranean deep-sea biota

Preliminary results of DNA barcoding survey of deep-sea mega-faunal biota are presented, collected by trawl and gillnet off the Israeli coast (SE Mediterranean, depths 700 to 1500 meters) during 2012-2013. 846 organisms were identified to 37 species, mainly fish and decapod crustaceans. The most abundant species were the blackmouth catshark Galeus melastomus, the cosmopolitan decapod Polycheles typhlops and the bivalve Abra longicallus. Two species were sampled for the first time from the southern Levant- the long armed chiroteuthid squid Chiroteuthis veranyi and the common mora, Mora moro. Four of the 18 fish species and two of the 10 crustacean species were abundant, representing 78% and 61%, respectively, of the organisms collected. Most other species are represented by fewer than 10 individuals. PCR products for the cytochrome c oxidase sub unit I (COI) gene for the 37 species were successfully sequenced. The identified and vouchered individuals are stored at the Steinhardt Museum of Natural History (Tel Aviv University, Israel) and their COI sequences were uploaded into the BoLD universal data center as part of the national marine barcoding project. The COI sequences of Acanthephyra eximia, Gryphus vitreus, Galeodea echinophora, Mesothuria intestinalis and Astropecten irregularis, constitute first records of these species in BoLD. When compared to the COI sequences in BoLD, the present results reveal some inconsistency in species identification, an outcome that should be taken into consideration primarily once the taxonomical verifications of collected taxa are elusive. This study is the first step in DNA barcoding of the Levant’s little-known benthic deep-sea fauna.

A Genetic Survey of Pyrethroid Insecticide Resistance in Aphids in New Brunswick, Canada, with Particular Emphasis on Aphids as Vectors of Potato virus Y.

Aphids are viral vectors in potatoes, most importantly of Potato virus Y (PVY), and insecticides are frequently used to reduce viral spread during the crop season. Aphids collected from the potato belt of New Brunswick, Canada, in 2015 and 2016 were surveyed for known and novel mutations in the Na-channel (para) gene, coding for the target of synthetic pyrethroid insecticides. Specific genetic mutations known to confer resistance (kdr and skdr) were found in great abundance in Myzus persicae (Sulzer) (Hemiptera: Aphididae), which rose from 76% in 2015 to 96% in 2016. Aphids other than M. persicae showed lower frequency of resistance. In 2015, 3% of individuals contained the resistance mutation skdr, rising to 13% in 2016 (of 45 species). Several novel resistance mutations or mutations not before reported in aphids were identified in this gene target. One of these mutations, I936V, is known to confer pyrethroid resistance in another unrelated insect, and three others occur immediately adjacent and prompt similar chemical shifts in the primary protein structure, to previously characterized mutations associated with pyrethroid resistance. Most novel mutations were found in species other than M. persicae or others currently tracked individually by the provincial aphid monitoring program, which were determined by cytochrome C oxidase I (cox1) sequencing. Through our cox1 DNA barcoding survey, at least 45 species of aphids were discovered in NB potato fields in 2015 and 2016, many of which are known carriers of PVY.

Insights into species diversity of associated crustose coralline algae (<em>Corallinophycidae, Rhodophyta</em>) with Atlantic European maerl beds using DNA barcoding

DNA barcoding in combination with morpho-anatomical analysis was applied to study the diversity of crustose coralline algae associated to two maerl beds from two protected Atlantic European areas from Brittany and Galicia —France and Spain, respectively—. Given the records of gametophytes of the maerl species Phymatolithon calcareum under crustose growth-forms, and that associated crustose coralline algae appear to be involved in the recruitment of new maerl plants, we compared the species composition between the associated crustose coralline algae to Breton and Galician maerl beds with the maerl species identified in these beds in previous DNA barcoding surveys. Our molecular results revealed higher species diversity in associated crustose coralline algae than in maerl-forming species. Nine taxa of crustose coralline algae were found in both study areas: four in Brittany and five in Galicia. Three species from Brittany were identified as Phymatolithon calcareum, Phymatolithon lamii, and Lithophyllum hibernicum. The remaining six ones were assigned to the genera Phymatolithon and Mesophyllum, along with Lithothamnion and Lithophyllum. Morpho-anatomical examination of diagnostic characters corroborated our molecular identification. Our results showed that the most representative genus of crustose coralline algae in Brittany was Phymatolithon, while in Galicia was Mesophyllum. In Brittany, Phymatolithon calcareum was found under both growth-forms, maerl and crustose coralline algae, the latter assigned to the gametophyte stage by the presence of uniporate conceptacles. The recruitment of new maerl plants involving associated crustose coralline algae with maerl beds may occur, but only we can affirm it for Phymatolithon calcareum in Brittany. By contrast, the different species composition between both growth-forms in the Galician maerl beds would indicate that the fragmentation of own free-living maerl species appears to be the most common propagation mechanism.

DNA in a bottle-Rapid metabarcoding survey for early alerts of invasive species in ports.

Biota monitoring in ports is increasingly needed for biosecurity reasons and safeguarding marine biodiversity from biological invasion. Present and future international biosecurity directives can be accomplished only if the biota acquired by maritime traffic in ports is controlled. Methodologies for biota inventory are diverse and now rely principally on extensive and labor-intensive sampling along with taxonomic identification by experts. In this study, we employed an extremely simplified environmental DNA (eDNA) sampling methodology from only three 1-L bottles of water per port, followed by metabarcoding (high-throughput sequencing and DNA-based species identification) using 18S rDNA and Cytochrome oxidase I as genetic barcodes. Eight Bay of Biscay ports with available inventory of fouling invertebrates were employed as a case study. Despite minimal sampling efforts, three invasive invertebrates were detected: the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus and the polychaete Polydora triglanda. The same species have been previously found from visual and DNA barcoding (genetic identification of individuals) surveys in the same ports. The current costs of visual surveys, conventional DNA barcoding and this simplified metabarcoding protocol were compared. The results encourage the use of metabarcoding for early biosecurity alerts.