DNA metabarcoding has the capacity to bolster current biodiversity assessment techniques, including the early detection and monitoring of non-indigenous species (NIS). However, the success of this approach is greatly dependent on the availability, taxonomic coverage and reliability of reference sequences in genetic databases, whose deficiencies can potentially compromise species identifications at the taxonomic assignment step. In this study we assessed lacunae in availability of DNA sequence data from four barcodes (COI, 18S, rbcL and matK) for NIS occurring in European marine and coastal environments. NIS checklists were based on EASIN and AquaNIS databases. The highest coverage was found for COI for Animalia and rbcL for Plantae (up to 63%, for both) and 18S for Chromista (up to 51%), that greatly increased when only high impact species were taken into account (up to 82 to 89%). Results show that different markers have unbalanced representations in genetic databases, implying that the parallel use of more than one marker can act complimentarily and may greatly increase NIS identification rates through DNA-based tools. Furthermore, based on the COI marker, data for approximately 30% of the species had maximum intra-specific distances higher than 3%, suggesting that many NIS may have undescribed or cryptic diversity. Although completing the gaps in reference libraries is essential to make the most of the potential of the DNA-based tools, a careful compilation, verification and annotation of available sequences is fundamental to assemble large curated and reliable reference libraries that provide support for rigorous species identifications.

BOLD, DNA barcode markers, gap-analysis, GenBank, marine and coastal ecosystems, non-indigenous species

Marine and coastal habitats are among the most important, but also the most threatened ecosystems in the world, providing invaluable services, such as provisioning, supporting, regulation and cultural/aesthetic for human well-being (Solan et al. 2004; Rilov and Crooks 2009). Along with climate change, habitat destruction, overexploitation and pollution, the spread of non-indigenous species (NIS), for areas outside their natural occurrence range, is among the five most important direct drivers of biodiversity loss in European coastal regions (MEA 2005). Due to its position as a centre for international trade over centuries, Europe has a large number and diversity of well-established NIS in marine and coastal habitats (Keller et al. 2011; Katsanevakis et al. 2013a,b, 2014; Tsiamis et al. 2019). Many of these species are, or can become, invasive and displace and out-compete native species, leading to severe ecological changes threatening ecosystem integrity (Molnar et al. 2008; Rilov and Crooks 2009; Simberloff et al. 2013). Impacts include community structure alterations, biodiversity loss, habitat modification, harm to human health and economic losses (Keller et al. 2011).

While morphology-based identification of taxa has largely ensured the ascertainment of the current status of NIS occurring in coastal environments in Europe (Keller et al. 2011; Katsanevakis et al. 2013a, 2014; Tsiamis et al. 2019), this process is expertise-demanding, laborious and time consuming. It is also hardly applicable to some poorly studied communities, such as interstitial fauna, which may have moved large distances in ships through ballast waters or sediments (Carlton 1999; Ojaveer et al. 2014; Shang et al. 2019; Shaw et al. 2019). Particularly in aquatic systems, an accurate identification and detection of NIS may be prevented by the presence of life stages not amenable to morphological identification (i.e. eggs, propagules, planktonic larvae, juveniles), or because organisms are not large and distinctive (e.g. meiofauna, microalgae, zooplankton, protists) (Pochon et al. 2015; Zaiko et al. 2015a, 2015b, 2015c, 2016; Pagenkopp Lohan et al. 2016, 2017) or occur in low abundances (Darling and Blum 2007). In the case of NIS, the accuracy of species identifications is paramount since incorrect identifications can lead to biased outcomes and action against harmless species or inaction against problematic ones (Briski et al. 2016; Viard et al. 2019).

DNA-based methods, such as DNA barcoding (Hebert et al. 2003) and DNA metabarcoding (Hajibabaei 2012; Cristescu 2014), offer great promise for reliable species identifications in invasive ecology, having considerable potential to overcome some of the above-mentioned challenges and to improve the monitoring of NIS in marine and coastal ecosystems (Briski et al. 2011, 2016; Zaiko et al. 2015a, 2015b, 2015c; Abad et al. 2016; Brown et al. 2016; Miralles et al. 2016, 2018; Holman et al. 2019; Wood et al. 2019; Rey et al. 2020). In particular, DNA metabarcoding, which allies amplicon barcoding with high throughput sequencing may have a number of potential benefits over traditional methods, including the simultaneous processing of a large number of samples and the simultaneous identification of multiple taxa from various types of environmental samples (Hajibabaei 2012; Taberlet et al. 2012; Shokralla et al. 2012; Cristescu 2014), increased sensitivity and specificity, often revealing hidden diversity (Lindeque et al. 2013; Viard et al. 2019), as well as greater time and cost effectiveness (Briski et al. 2011, 2016; Pochon et al. 2015; Brown et al. 2016; von Ammon et al. 2018; Holman et al. 2019; Rey et al. 2020). In addition, a species may be detected at early developmental stages and before its dispersal and impact become readily apparent and irreversible (Pochon et al. 2015; Holman et al. 2019).

Efficient and accurate species identifications through DNA barcoding or DNA metabarcoding are dependent on reliable reference sequences libraries of known taxa (Briski et al. 2016; Miralles et al. 2016, 2018; Viard et al. 2019; Weigand et al. 2019). The unavailability or under-representation of some taxonomic groups in genetic databases may lead to biased results in biodiversity assessments through DNA-based tools and restrict the resolution and detection capacity of NIS at the taxonomic assignment step (Pochon et al. 2015; Briski et al. 2016; Chain et al. 2016; Zaiko et al. 2016; Lacoursière-Roussel et al. 2018; von Ammon et al. 2018; Rey et al. 2020). In Europe, comprehensive and reliable barcode reference libraries would be mandatory if DNA-based tools are to be implemented in biomonitoring in the context of the European Water Framework Directive (WFD, Directive 2000/60/EC) and the Marine Strategy Framework Directive (MSFD, Directive 2008/56/EC) (Leese et al. 2016, 2018; Hering et al. 2018; Pawlowski et al. 2018; Weigand et al. 2019). However, no recent attempt has been made for assessing the gaps in NIS sequences in publicly accessible databases (i.e. the number of species missing barcode sequences). To our best knowledge the most recent complete gap-analysis was conducted in 2016 (Briski et al. 2016) and although a recent one was performed in 2019 (Ardura 2019), it targeted only high-impact Arthropoda and Mollusca species.

In the current study, the gaps, for the most commonly used barcode markers in DNA-based studies for Animalia (COI and 18S), Chromista (COI, 18S and rbcL) and Plantae (COI, rbcL and matK), were analysed in the genetic databases GenBank and the Barcode of Life Data System (BOLD), with a focus on NIS occurring in European coastal regions by using updated lists retrieved from the European Alien Species Information Network (EASIN) (Katsanevakis et al. 2012) and the Information System on Aquatic Non-indigenous and Cryptogenic species (AquaNIS) (Olenin et al. 2014). This will allow a current appraisal of the status of NIS occurring in European marine and coastal regions that are missing DNA barcodes, and will permit researchers to develop actions to fulfil these gaps, in order to take the most of the potential of NIS identification through DNA-based tools. Actions developing innovative tools for biodiversity monitoring are mandatory for an effective management of biological invasions and the development of mitigation strategies to deal with increasing globalization and environmental change.

Our study brings to the forefront two main considerations: first, reference libraries still lack representative sequences for many NIS with extreme cases in some groups, and second, some NIS can be categorised as possible cryptic species. Both these cases may critically impair the detection of NIS and therefore, the current capability for NIS detection and monitoring using molecular tools.

Although the gaps (i.e., NIS still missing barcode sequences) were similar in both lists, the values of missing barcodes clearly differed among taxonomic groups and the barcode markers searched. In both lists the gap was highest for Chromista. In these lists, Chromista include Foraminifera, Myzozoa and Ochrophyta as dominant phyla, that can harbour very small sized species, such as small protists and diatoms and for which obtaining voucher specimens to generate sequences to deposit in genetic databases may be challenging. It has been reported that smaller organisms may have greater invasion opportunities in coastal ecosystems (Ruiz et al. 2000; Pagenkopp Lohan et al. 2016, 2017), but that can be hard to detect by using traditional morphological approaches (Pagenkopp Lohan et al. 2016, 2017). Thus, DNA-based tools are essential for its early detection and accurate identification in recipient ecosystems and fulfilling the gaps in barcode reference libraries is extremely essential for Chromista.

On the other hand, we found a lower gap for Animalia and Plantae. The gaps in BOLD and GenBank were recently analysed for the taxa frequently used in the WFD and the MSFD, under the scope of the COST Action DNAqua-Net (Weigand et al. 2019), and the authors also found that barcode coverage varied strongly among taxonomic groups. In general, groups that were actively targeted in barcode projects were well represented in the barcode libraries, while others have fewer records. Our results support this trend. Under the scope of the public project “WG1.8 Marine Bio-Surveillance” deposited in BOLD, 12 and 17 projects were dedicated to Animalia and Plantae, respectively, with a total of 1,516 sequences, while only 4 projects were dedicated to Chromista, comprising only 105 sequences. In both lists, the phyla with the highest number of records in the two searched genetic databases include a high number of species having a high impact in the environment or species with high economic value (i.e. Chordata, Arthropoda, Mollusca, Ochrophyta, Rhodophyta). These species are generally the focus of a greater number of studies and thus, may display a higher trend of sequence deposition in genetic databases (Briski et al. 2011, 2016; Pyšek et al. 2008; Trebitz et al. 2015; Ardura 2019). In fact, we found among the top ten species with the highest number of sequence records either high impact species, such as Callinectes sapidus and Anguillicoloides crassus, or species with high economic value such as Mytilus trossulus, Prionace glauca, Cyprinus carpio and Oncorhynchus mykiss.

Our results were somewhat discrepant from those obtained in a previous report where the gaps in BOLD and GenBank were analysed for aquatic NIS compiled from literature (Briski et al. 2016). By 2016, 76% of the species in the list, compiled by Briski and colleagues for aquatic NIS (n=1,383), had at least one sequence of 6 searched markers in BOLD or GenBank. In addition, the authors predicted that if the rate of sequence deposition in both genetic databases followed a linear trend, they would expect that all aquatic NIS in their list would be sequenced by 2030. In our study, completion seems to be still a bit far off with only 65% of the species in the AquaNIS and 55% in the EASIN list having at least one of the searched barcode markers in BOLD or GenBank. These disparities probably originated from different compliance criteria and mismatching of the species lists used in the analyses, which in the case of Briski and colleagues (2016) consisted on a list of NIS occurring at a worldwide scale. In addition, only barcode sequences higher than 500 bp were considered in the current gap analysis, while Briski et al. (2016) did not mention if any length filter has been applied to their sequences search. New NIS and new introductions into different recipient regions are reported every year and NIS status can also change (from unknown status to cryptogenic or alien), suggesting that this is a work that needs to be performed from time to time. Fortunately, currently, there are specific databases dedicated to this, and that are constantly updated, such as EASIN and AquaNIS (Katsanevakis et al. 2012; Olenin et al. 2014), which greatly facilitates this task. In addition, the R-based bioinformatic pipeline, developed in our study to retrieve the information relative to each marker from the two genetic databases, will enable to conduct this task effectively and in an automated way when needed (i.e. every time that significant updates are made in the lists).

As above-mentioned, for each taxonomic group, the gap clearly differed among the barcode markers searched. For Animalia, most phyla were well represented with COI sequences in GenBank and BOLD, but Annelida, Ctenophora, Platyhelminthes and Porifera were better represented with 18S sequences. Within Chromista most phyla were better represented with 18S, but for instance Ochrophyta, which includes brown algae and diatoms, was an exception to this pattern, with the barcode coverage being greatest for rbcL. For Plantae, most phyla were better represented with rbcL sequences. Thus, the simultaneous use of more than one marker can act complimentarily and may greatly increase NIS identification rates through DNA-based tools. Recent studies have highlighted the advantage of using both 18S and COI markers for invasive species detection; the 18S for detecting a much broader range of taxa and the COI for discriminating between many metazoan species (Borrell et al. 2017; von Ammon et al. 2018; Stefanni et al. 2018; Holman et al. 2019; Wood et al. 2019; Rey et al. 2020). In addition, the concomitant use of the rbcL and COI allowed the detection of diatoms and green and yellow algae, in ballast water of a vessel crossing the Atlantic Ocean, which otherwise would remain highly underestimated if communities have been only targeted with COI (Zaiko et al. 2015b).

Approximately 37% of the species displayed more than one BIN, and many of these species displayed mean- and maximum-intraspecific distances higher than 3%, suggesting that many NIS may display hidden diversity or cryptic diversity, which may further complicate taxonomic assignment using DNA-based tools (Viard et al. 2019). In addition, many species were represented by singletons in the genetic databases, thereby preventing detection of possible intraspecific variability or cryptic diversity. At the moment, at least to our knowledge, no dedicated reference sequences database exists for NIS. Ideally, and also suggested by the great proportion of species displaying multiple BINs and high intraspecific distances in the current study, this reference database should cover the full sweep of species in the target ecosystem, with a balanced representation of specimens across each species distribution range in both native and recipient locations, to account for the possible regional variability in targeted barcode genes. In addition, database incompleteness can be somewhat overcome by the addition of DNA sequences for local species. Abad et al. (2016) was able to increase 2 times more the success of the taxonomic assignment of plankton species in the estuary of Bilbao (Spain), by generating DNA barcodes for local species before conducting a metabarcoding-based study.

A closer look at the list of barcoded species with attributed BINs, in particular for COI and Animalia, indicated that many of them displayed discordant BINs (i.e. different species sharing the same BIN), possibly due to incorrect taxonomic assignments of numerous species, that have been repeatedly used in databases without a proper validation. A careful inspection in these BINs would be needed in order to check for potential artefacts such as misidentifications, incomplete taxonomy or sequences that were deposited under different synonyms. Incorrect species identifications could either artificially inflate or depress the number of NIS in an ecosystem, and lead to misdirecting limited resources against harmless species or inaction against problematic ones (Bax et al. 2001; Simberloff 2009). Lacoursière-Roussel et al. (2018) identified Acartia tonsa through eDNA metabarcoding, in water samples collected at two Canadian ports, a potential invader that has been previously recorded in the ecoregions of ports connected to Churchill. However, the current available COI sequences for A. tonsa form several distinct clades, some of which cluster with A. hudsonica, which rose the possibility that the eDNA sequences assigned to A. tonsa may belong to the native A. hudsonica. Very recently, by examining public databases Viard et al. (2019) also found sequences of Botrylloides diegensis erroneously assigned to B. leachii. This observation has major implications as the introduced B. diegensis can be misidentified as a putatively native species. Unfortunately, these database errors can be frequent, as also suggested by the high proportion of discordant BINs found in the current study, and can delay the implementation of DNA metabarcoding in NIS surveillance in coastal ecosystems.

Although completing the gaps in reference libraries is essential to make the most of the potential of DNA-based tools in NIS surveillance in coastal ecosystems, correct species attribution (by morphology-based methods) and proper management of sequence deposition and voucher storage is vital to preserve correct connections between morphological and molecular data (Briski et al. 2016). This can be particularly challenging for small-sized species that lack unambiguous morphological traits to use in taxonomic diagnosis, such as some particular groups within Chromista (e.g. Myzozoa), for which a higher gap was found in genetic databases. In addition, a careful compilation, verification and annotation of each database record is fundamental to assemble large, curated and reliable reference libraries that provide support for rigorous species identifications through DNA-based tools (Viard et al. 2019; Weigand et al. 2019; Fontes et al. 2020; Leite et al. 2020). This need is particularly acute for the phylogenetically diverse NIS, for which there is highly dispersed data that needs to be compiled and verified. Once this need is fulfilled, the adoption of DNA-based tools for accurate NIS detection and monitoring in marine and coastal ecosystems will very likely accelerate.

This work was supported by national funds through the Portuguese Foundation for Science and Technology (FCT, I.P.) in the scope of the project “NIS-DNA: Early detection and monitoring of non-indigenous species (NIS) in coastal ecosystems based on high-throughput sequencing tools” (PTDC/BIA-BMA/29754/2017). We are also grateful to two reviewers for comments and suggestions that improved the manuscript.