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Research Article
First joint morphological and molecular detection of Watersipora subatra in the Mediterranean Sea presented in an updated genus phylogeny to resolve taxonomic confusion
expand article infoRobin P. M. Gauff‡§, Marc Bouchoucha§, Amelia Curd‡, Gabin Droual‡|, Justine Evrard‡, Nicolas Gayet, Flavia Nunes‡
‡ IFREMER, DYNECO, Laboratory of Coastal Benthic Ecology, Plouzané, France
§ IFREMER, Lab Environm Ressources Provence Azur Corse, La Seyne Sur Mer, France
| IFREMER, INRAE, Institut Agro—Agrocampus Ouest, Ecosystem Dynamics and Sustainability, Nantes, France
¶ CNRS, IFREMER, UBO, Biology and Ecology of Deep-Sea Ecosystems, Plouzané, France

Corresponding author: Robin P. M. Gauff ( gauff.robin@yahoo.de )

Academic editor: Kevin C.K. Ma
© 2023 Robin P. M. Gauff, Marc Bouchoucha, Amelia Curd, Gabin Droual, Justine Evrard, Nicolas Gayet, Flavia Nunes.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Gauff RPM, Bouchoucha M, Curd A, Droual G, Evrard J, Gayet N, Nunes F (2023) First joint morphological and molecular detection of Watersipora subatra in the Mediterranean Sea presented in an updated genus phylogeny to resolve taxonomic confusion. Aquatic Invasions 18(3): 295-312. https://doi.org/10.3391/ai.2023.18.3.108128
Open Access

Abstract

Introduced species constitute a critical bio-security issue worldwide and the precise monitoring of their spread is crucial for their management. For species forming cryptic complexes this may remain difficult. Using integrative taxonomy, we formally report for the first time, well-established populations of the cosmopolitan introduced bryozoan Watersipora subatra in the French Mediterranean Sea and compile worldwide existing genetic data for Watersipora species alongside newly acquired data to establish the most complete phylogeny of the genus to date. This revealed pervasive erroneous identifications in Genbank, which in turn perpetrate further errors in recent studies, primarily misidentifying W. subatra as W. subtorquata. High abundance and geographic spread of W. subatra in our Mediterranean sampling sites suggest that this species has been present for some time but has been misidentified until now. We provide an updated species identification for all current reference sequences in the Watersipora genus, which may help future monitoring of W. subatra and other Watersipora species.

Key words

Bryozoa, integrative taxonomy, introduced species, phylogeny, NIS

Introduction

Marine interconnectivity among nations has risen in recent decades, a trend that will further increase and that is favoring species introductions all over the world (Levine and D’Antonio 2003; Seebens et al. 2016; Carrasco et al. 2017; Sardain et al. 2019). As introduced species lack natural regulators in their new environment (Papacostas et al. 2017), their effects may be unpredictable. Some invaders may completely restructure ecosystems, potentially leading to the loss of biodiversity and ecosystem services (Pejchar and Mooney 2009; Johnston et al. 2015; Walsh et al. 2016), causing local extinctions (Blackburn et al. 2019), and high economic impacts (Lovell et al. 2006; Olson 2006; Jardine and Sanchirico 2018; Diagne et al. 2021). As an example, direct damages as well as costs generated from combating introduced species accumulated to over $29 billion in Europe alone (see supplementary material table S1 of Diagne et al. 2021). These damages on ecosystems and the economy do not even require high abundances of an introduced species to be tangible (Blackburn et al. 2011). For these reasons, invasive species (sensu Blackburn et al. 2011) are regarded as a crucial global biosecurity issue, and prevention and early management of these species constitutes the best strategy to minimize their impact (Lovell et al. 2006; Olson 2006; Pyšek et al. 2020). This may prove difficult in phyla with few or small morphological identification criteria, that form cryptic species complexes (Mackie et al. 2006; Mackie et al. 2012; Vieira et al. 2014; Mastrototaro et al. 2020; Salonna et al. 2021), or for which taxonomic expertise is rare, such as for many marine invertebrates. Identifying the precise species at a certain location is however important for subsequent evaluation of spread and invasion (Vieira et al. 2014; Golo et al. 2023).

Especially when morphological criteria are lacking to identify a species, genetic methods have been increasingly used to help with the identification of introduced species. DNA barcoding of individual specimens, metabarcoding of communities in bulk (from sediments, scrapings, or other substrates) and more recently environmental DNA (eDNA) are now all regarded as useful techniques for detecting introduced species. However, the efficiency of molecular species identification highly depends on the quality of reference sequences, particularly how well species identification was carried out for the reference sequences before being submitted to public databases (Couton et al. 2022). Incorrect species identification of reference sequences can have important effects on subsequent studies which will base molecular identifications on imprecise species names.

The morphology of the bryozoan genus Watersipora is notoriously complicated, and the redescription of the genus by Vieira et al. (2014) has reattributed individuals of introduced populations in multiple localities to Watersipora subatra (Ortmann, 1890) (identified as “Watersipora subtorquata” (d’Orbigny, 1852) in Mackie et al. 2012; Ryland et al. 2009) and others to W. subtorquata (identified as “W. subvoidea” (d’Orbigny, 1852) in Mackie et al. 2006, 2012). This has led to confusion regarding the identity of populations of several localities around the world, most notably in Europe. Many species identifications of reference sequences in Genbank have not been updated since the redescription by Vieira et al. (2014). Nevertheless, these sequences continue to be used for e-DNA monitoring. As an example, individuals that were resolved as W. subatra are still listed as “W. subtorquata” on GenBank (accessed June 2022, NCBI, Benson et al. 2013). This probably induces subsequent errors in the literature, falsely detecting W. subatra as “W. subtorquata”, hindering the accurate e-DNA detection of several Watersipora species.

In the Mediterranean Sea, the presence of W. subtorquata has been reliably confirmed (Vieira et al. 2014) and this species is the most frequently described introduced Watersipora species in this area (Vieira et al. 2014; Harmelin et al. 2016; Rosso and Di Martino 2016; Tempesti et al. 2020), even though Watersipora arcuata Banta, 1969 seems to be spreading rapidly in recent years (Ulman et al. 2017, 2019; Reverter-Gil and Souto 2019). Here however, we identified W. subatra as the dominant Watersipora species in several French Mediterranean harbors, with only anecdotal presence of W. subtorquata (and no observations of W. arcuata). This points towards an inherent identification problem, as the high abundance and persistence on many artificial substrates makes it impossible to miss the species (see Fig. 1a) and suggests it has been present some time already.

Figure 1. 

Living Watersipora subatra colony from the Toulon Bay in situ (A) (Benoist de Vogüé/IFREMER) and under optic microscope (B). Opercula with a dark central band and swirls are visible (Robin Gauff).

The present study has a two-fold objective. Firstly, we wish to declare the first formal record of W. subatra as an already well-established introduced species in the French Mediterranean Sea. Secondly, we provide a phylogenetic analysis of existing COI sequences of Watersipora from Genbank, including new sequences from individuals that were carefully identified according to morphological criteria, in order to improve molecular identification and detection of non-indigenous Watersipora species, particularly the spread of W. subatra in the Mediterranean or elsewhere.

Materials and methods

Study area

Specimens for this study were sampled in four different locations along the French Mediterranean coastline. Three sample sites were under pontoons and docks in the Toulon Bay: in front of the Ifremer facilities (43.105415°N, 5.885415°E), in the La Seyne sur Mer marina (43.102007°N, 5.882377°E), and in the Toulon Darse Nord marina (43.114637°N, 5.931267°E), as well as a fourth site in the Old Harbor of Marseilles (43.293622°N, 5.363857°E). The Toulon Bay is a highly urbanized area (Meaille and Wald 1990), with six marinas, several commercial harbors, a large military harbor and ferry activities over an area of approximatively 10 km². It is highly impacted by anthropogenic pressures such as habitat modification and loss (Bouchoucha et al. 2016, 2018a, b), chemical contamination (Wafo et al. 2016; Araújo et al. 2019; Mazoyer et al. 2020), and the presence of introduced species (Zibrowius 1991; Ruitton et al. 2005; Gauff et al. 2023a)). The Old Harbor of Marseilles is a recreational marina with approximately 3200 boat moorings, a large hull cleaning area, and commercial activities such artisanal fisheries and short distance ferry transports. Being the second largest French city and having a large harbor complex 7.5 km², and with more than 10 km of continuous artificial coast, Marseilles constitutes another key example of massive marine urbanization in the Mediterranean Sea. Tentative identifications have noted W. subatra as being present in this area from 2019 on (Gauff et al. 2023b)), however a detailed morphological description and completely reliable analysis was until now lacking. In order to compare Mediterranean individuals with well-known W. subatra populations (Ryland et al. 2009), we sampled additional colonies at a fifth site at the Pointe du Diable close to Brest, Brittany, France (48.354768°N, 4.558518°W) where W. subatra seems until now to be the only representative species of the genus (Leclerc and Viard 2017; Porter et al. 2017; Gauff et al. 2022).

Morphological analysis

Approximately 200 g of Watersipora colonies from the studied locations were sampled for the present study and scanned for different species. Individuals were first identified alive in the laboratory using a ZEISS SteREO Discovery.V12 microscope coupled to a ZEISS Axiocam 506 mono camera and visualized and measured with ZEISS Zen 3.0 software. Operculum structure (see figures 65–68 in Vieira et al. 2014) and general individual characteristics (Zooid Length, Zooid Width, Orifice Length, Orifice Width, Sinus Length, Sinus Width, Pseudopore Diameter, Intrazooidal Septula Presence and Diameter; see tables 1, 3 in Vieira et al. 2014) were used as first identification criteria. A total of 15 high-quality fragments (no epibionts, not epibionts themselves, clean, alive…) were chosen for detailed analysis and description (3 for each of the three areas of the Toulon Bay, 3 for Marseilles and 3 from Brittany). The colonies were then prepared for scanning electron microscopy (SEM). Specimen fragments were bleached for 48 hours, washed in deionized water, then dried at 37 °C overnight. Clean fragments were mounted on stubs with carbon glue, and sputter-coated with 60%Au/40%Pd. Images were taken with 200×, 600× and 2500× magnification with a FEI Quanta 200 SEM. The targeted identification criteria here were the latero-oral intrazooidal septula (IZS) which allow to clearly distinguish W. subatra (IZS present) from W. subtorquata and W. souleorum Vieira, Specer Jones & Taylor, 2014 (IZS absent; Vieira et al. 2014). Measurements of intrazooidal septula and pores were taken with the ImageJ (Rueden et al. 2017) ‘Analyse’ tool using the scale from SEM images. The remaining colony was immediately preserved in absolute ethanol for genetic sequencing.

DNA extraction, amplification and sequencing

Zooids were removed from their epitheca to avoid contamination with exogenous DNA. Twenty zooids were pooled per colony, and DNA was extracted using the NucleoSpin DNA RapidLyse kit (Macherey-Nagel) following the manufacturer’s protocol. Polymerase chain reaction (PCR) of the mitochondrial cytochrome c oxidase I gene was conducted with primers designed specifically for Bryozoa: BryCOIL1548 forward 5'- CAT AAC AGG AAG AGG TTT AAG -3' and BryCOIH2161 reverse 5'- ATY AGG AGC AGG ATT CAG TAT G -3' (Mackey et al 2006). PCR amplifications were performed in a total volume of 25 µl with the DreamTaq DNA polymerase (ThermoFisher), consisting of 2.5 µl DreamTaq PCR Buffer (10×, including 20 mM MgCl2), 0.5 µl dNTPs (10 mM each), 1 µl of each primer (10 µM each), 0.2 µl of DreamTaq polymerase, 17.8 µl sterile Millipore water, and 2 µl of DNA. The thermal cycling protocol included an initial denaturation step at 94 °C (3 min), followed by 35 cycles including denaturation at 94 °C (30 s), annealing at 50 °C (30 s), and elongation at 72 °C (60 s). The PCR products were run through a 1% agarose gel prepared with Tris-borate EDTA (TBE). Two bands were observed in the PCR product. The 650 bp fragment was excised from the gel and was purified using the Nucleospin Gel and PCR Clean Up kit (Macherey-Nagel). Sanger sequencing was conducted at Eurofins Genomics in both forward and reverse directions.

Phylogenetic analysis

Sequence chromatograms were trimmed for low quality bases and visually inspected for errors in ‘Genieous Prime’ (v.2020.2.4; Dotmatics). Forward and reverse fragments were aligned to generate a consensus sequence. High quality sequences were obtained for three individuals from Marseilles, two individuals from Toulon and three individuals from Brest. They were deposited on GenBank (NCBI; Benson et al. 2013), under the accession numbers OQ918440–OQ918447. All sequences attributed to the genus Watersipora were downloaded from ‘GenBank’ (accessed on 01/11/2022) by using the search term “Watersipora” in the Nucleotide search engine. This returned 264 sequences from which we chose only COI sequences (229). Two sequences of W. platypora Seo, 1999 were excluded from our database as they were identical, and one sequence from eDNA detection from Portas et al. (2022) was added. Finally, we added eight sequences acquired for the present study. An alignment was generated for a total of 236 individuals using ‘Sequencher’ (v.5.3; Gene Codes Corp). The alignment was then trimmed manually to remove sections with high levels of missing data in the 5' and 3' ends. Identical sequences were removed from the dataset using ‘DAMBE’ (v 7.5.3; Xia 2018), to generate a non-redundant dataset composed of 99 unique sequences. A re-alignment of this final dataset was conducted with ‘MAFFT’ (v7.490; Katoh and Standley 2013), using the ‘--localpairs’ algorithm and a maximum number of 1000 iterations. Models of sequence evolution were tested with ‘modeltest-ng’ (v0.1.6; Darriba et al. 2020), and the model with the highest probability score was selected by considering the Bayesian information criterion (BIC), Akaike information criterion (AIC) and the corrected AIC (AICc). Maximum likelihood phylogenetic analysis was conducted with ‘iqtree’ (v2.0.3; Minh et al. 2020) using the HKY+G4 model and 1000 ultra-fast bootstrap replicates (Minh et al. 2013). Bayesian phylogenetic analysis was conducted with ‘BEAST’ (v1.10.4; Suchard et al. 2018), using a strict clock and the HKY model of sequence evolution with 4 gamma categories of site heterogeneity. The proportion of invariant sites and base frequencies were estimated, and the Yule process of speciation model was used, using default priors for all estimated operators. Three independent runs were conducted over 107 generations sampled every 1000 iterations. The logs for each run were examined to ensure an adequate effective sample size (ESS) had been reached for each estimator. The logs of the three runs were combined using ‘logcombiner’, and trees were summarized with ‘TreeAnnotator’ in ‘BEAST’, using maximum clade credibility and median branch heights, and a burn-in, i.e., the number of samples to be discarded at the start of the run, of 6000 trees (20%). Phylogenetic trees were visualized with ‘FigTree’ (v1.4.4; Rambaut 2010) and rooted on the W. arcuata clade. All datasets, tree files and the code of our analyses can be consulted at https://gitlab.ifremer.fr/lebco/fnunes/watersipora.git.

Sequence accession numbers were color coded according to the species identification indicated in the Genbank record. For clarity, in this manuscript identifications from Genbank will appear in double quotation marks (ex: “W. subtorquata”) as some were already resolved to other species by Vieira et al. (2014). Exceptions to this are proven reliable identifications (ex: Couton et al. 2019; McCann et al. 2019). For redundant sequences, only one accession number was listed per species name on the phylogenetic tree, with the number of individuals having an identical sequence indicated in brackets, however a detailed phylogenetic tree can be accessed in the Suppl. material 1: table S1.

Watersipora distribution map

We used the data obtained from our genetic sequencing and recent records of Watersipora spp. (Mackie et al. 2006; Anderson and Haygood 2007; Knight et al. 2011; Mackie et al. 2012; Porter et al. 2015; Ulman et al. 2017; Aleman et al. 2018; McCann et al. 2019; Reverter-Gil and Souto 2019; Ramalho and Caballero-Herrera 2022) to complement the map (figure 72 in Vieira et al. (2014).

Results

Morphological analysis

The characteristics from all 12 sampled individuals, allowing each to be identified as W. subatra using Vieira et al. (2014), were as follows: Zooid length 1118 ± 178 µm; Zooid width 476 ± 132 µm; Orifice skull-shaped with condyles; Orifice length (Zooidal plane) 263 ± 27 µm; Orifice width 318 ± 25 µm; Sinus U shaped; Sinus length (depth) 72 ± 13 µm; Sinus width 155 ± 14 µm (Table 1); Operculum with distinct central band and two clearer swirls (Fig. 1b); Latero-oral intrazooidal septula present (Fig. 2G–I); Pseudopore diameter 24 ± 5 µm; Condyles bar-shaped. Slight differences were observed between individuals from the Toulon Bay and the Marseilles Old Harbor. Toulon individuals were slightly larger, however, their Latero-oral intrazooidal septula were most often smaller than pseudopores, while they were larger than pseudopores for Marseilles individuals (Table 1). During a 3 h scan of the 200 g of sampled colonies, no other species than W. subatra could be identified.

Figure 2. 

Electron microscope photographs of Watersipora subatra colonies from Toulon, Marseilles, and Brest (A–C), their orifices with more detail (D–F) as well as their intra-zooidal septula, orifice margin and condyles (G–I) of the three sampled areas (columns) (Nicolas Gayet).

Table 1.
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Dimensions of the zooids (Mean ± SD in µm) of Watersipora subatra in the sampled areas. ZL: Zooid Length; ZW: Zooid Width; OL: Orifice Length; OW: Orifice Width; SinL: Sinus Length; SinW: Sinus Width; PorD: Pseudopore Diameter; IZSD: Intrazooidal Septula Diameter.

Toulon Bay Marseille Total Mediterr. Atlantic
Mean ± SD Mean ± SD Mean ± SD Mean ± SD
ZL 1179 ± 137 935 ± 166 1118 ± 178 878 ± 26
ZW 488 ± 134 441 ± 132 476 ± 132 430 ± 71
OL 270 ± 20 242 ± 35 263 ± 27 221 ± 17
OW 323 ± 21 301 ± 31 318 ± 25 298 ± 57
SinL 70 ± 14 75 ± 9 72 ± 13 46 ± 10
SinW 153 ± 13 163 ± 15 155 ± 14 138 ± 25
PorD 24 ± 5 25 ± 4 25 ± 4 23 ± 4
IZSD 25 ± 5 36 ± 6 28 ± 7 19 ± 6

Phylogenetic analysis

The alignment used for phylogenetic analysis contained 99 unique sequences and was 493 base pairs long. Of the 44 models of sequence evolution tested in ‘Modeltest-ng’, the HKY+G4 model had the highest lnLikelihood using BIC and AICc, while the TVM+G4 model had the highest lnLikelihood using AIC. Given the agreement between BIC and AICc, HKY+G4 was selected. Phylogenetic relationships were similar between trees produced with maximum likelihood and Bayesian methods. The phylogenetic tree obtained with the combined results of three independent runs on ‘BEAST’ is shown in Fig. 3. An extended version of this figure as well as all retrieved sequences can be found in the supplementary material (Suppl. materials 1, 3: table S1 and fig. S1). Nodes were annotated with posterior probabilities from the ‘BEAST’ analysis followed by the ultra-fast bootstrap values from ‘iqtree’, both expressed as percentages (i.e., 100/100).

Figure 3. 

Combined Bayesian (BEAST) and Maximum likelihood (iqtree) phylogenetic tree of all Watersipora COI sequences available on Genbank (With accession number and corresponding source references). Numbers at the nodes correspond to the posterior probabilities from the ‘BEAST’ analysis followed by the ultra-fast bootstrap values from ‘iqtree’, both expressed as percentages (i.e., 100/100). Only one accession number was listed per species for redundant sequences (number identical of sequences in parenthesis). New sequences acquired in this study are indicated by *. Color coding refers to species names listed on Genbank. Corrected identification (See Suppl. materials 1, 3: table S1 and fig. S1) are: Clade 1 W. arcuata; Clade 2 W. subtorquata; Clade 3 “Watersipora sp. sensu Mackie et al., (2012)”; Clade 4 “W. edmondsoni”; Clade 5 W. subatra.

Clade 1 formed a monophyletic group with strong support (100/100). All sequences were attributed to W. arcuata from three different studies, with samples from Australia, California and Hawaii (Mackie et al. 2006, 2012; Anderson and Haygood 2007) grouped in this clade. Two sequences previously attributed to “W. subtorquata” (Mackie et al. 2012) were also clustered within this group. Four additional clades were also found to have strong support values (ranging from 100/97 to 100/100), presumably corresponding to species level distinctions.

Clade 2 included the sequences for W. subtorquata sensu Vieira et al. (2014) from Galapagos (McCann et al. 2019), “W. subovoidea” from Brazil, Florida and Australia (Mackie et al. 2006; Mackie et al. 2012) and unidentified Watersipora sp. from California, Australia (Susick et al. 2020) and Washington (Mackie et al. 2012).

Clade 3 included sequences attributed to a potentially undescribed species of Watersipora sp. from California (Mackie et al. 2006, 2012) and “W. subtorquata” from Korea (Lee et al. 2011) and California (Anderson and Haygood 2007; Mackie et al. 2014).

Clade 4 included two sequences attributed to “W. edmondsoni” Soule & Soule, 1968 from Hawaii (Mackie et al. 2006; Mackie et al. 2012), a species currently considered invalid and synonymous with W. subtorquata (Vieira et al. 2014).

Clade 5 is comprised of sequences attributed to W. subatra sensu Vieira et al. (2014) from the Atlantic (Couton et al. 2019), unidentified Watersipora sp. from Australia and California (Susick et al. 2020) “W. subtorquata” from Australia (Mackie et al. 2006), California (Anderson and Haygood 2007; Mackie et al. 2012, 2014; Suppl. material 1: table S1 MK550661), Korea (Lee et al. 2011), New Zealand (Knight et al. 2011), Spain (Miralles et al. 2018), the United Kingdom (Ryland et al. 2009) and the French Mediterranean and French Atlantic (Ryland et al. 2009; Portas et al. 2022). This clade also contained our W. subatra samples from the French Atlantic and French Mediterranean. Clade 5 can itself be subdivided into two sub-clades (Clade 5.A and Clade 5.B; node 4) which correspond to the two clades previously identified in Mackie et al. (2012; as “W. subtorquata”).

Phylogenetic relationships among the clades 2–5 indicate that clades 4 and 5 (“W. edmondsoni” and W. subatra) are sister taxa grouped into node 3 (96/76). “Watersipora sp. sensu Mackie et al. (2012)” is grouped with node 3 into node 2, although support values were low for this node (83/76). Finally, W. subtorquata was sister to node 2 containing “W. edmondsoni”, W. subatra and “Watersipora sp. sensu Mackie et al. (2012)” clade (100/100; node 1).

Watersipora spp. distribution map

The new distribution map of Watersipora spp. (Fig. 4; Suppl. material 2: table S2) adds two species identities (“W. edmondsoni” and “Watersipora sp. sensu Mackie et al. (2012)”) and removes one (“W. complanata” now Terwasipora complanata (Norman, 1864)) compared to Vieira et al. (2014): figure 72). The updated map includes new records of W. subatra in Washington state, United States of America, and in the Mediterranean identified through the phylogeny and our samplings. This map extends records of Watersipora spp. to regional occurrence scales (W. subatra has for instance been shown to occur throughout S-E Australia). New reliable records of W. subatra in Norway and the Iberian Peninsula (Porter et al. 2015; Reverter-Gil and Souto 2019; Ramalho and Caballero-Herrera 2022), new records of W. subtorquata in Galapagos (McCann et al. 2019), as well as new records of W. arcuata throughout the Mediterranean (Ulman et al. 2017) are also included.

Figure 4. 

Distribution map of Watersipora spp. completing Vieira et al. (2014; fig. 72.) with updated species occurrences and identities based on genetic sequences, and recent reports (Porter et al. 2015; Ulman et al. 2017; McCann et al. 2019; Reverter-Gil and Souto 2019; Ramalho and Caballero-Herrera 2022; See also Suppl. material 2: table S2).

Discussion

At least five species of Watersiporidae have been reported from the Mediterranean Sea, including Watersipora cucullata (Busk, 1854), W. souleorum, Terwasipora complanata and the introduced W. subtorquata and W. arcuata (Vieira et al. 2014; Ulman et al. 2017; Reverter-Gil and Souto 2019). Watersipora subatra, despite being the most dominant introduced Watersiporidae in the north-eastern Atlantic, has only been recorded recently and sporadically in the Mediterranean Sea (Fernández-Romero et al. 2021; Ramalho and Caballero-Herrera 2022; Gauff et al. 2023a, b)). The individuals examined within our study unambiguously correspond to the morphological description of W. subatra in Vieira et al. (2014). This is further validated by the genetic analysis that cluster our Mediterranean individuals within the clade regrouping individuals of “W. subtorquata” from Mackie et al. (2006, 2012), that were resolved as W. subatra (Vieira et al. 2014), individuals identified as W. subatra from the French Atlantic (Couton et al. 2019) and our own W. subatra from the French Atlantic. We thus can confidently report the presence of this species in the French Mediterranean Sea. Out of the 13 accepted Watersipora spp., all four species that are spreading throughout the world (W. subtorquata, W. souleorum, W. arcuata and W. subatra) are thus now present and established in the Mediterranean Sea. Watersipora subatra is an invasive fouling species, mostly dispersed by ship traffic, that has recently spread from the north-east Atlantic towards the south of the Iberian Peninsula, suggesting that its introduction into the Mediterranean Sea likely occurred through the straits of Gibraltar (Reverter-Gil and Souto 2019). France possesses an Atlantic and Mediterranean coast, harboring numerous introduced species (Massé et al. 2023). National commerce and exchange (such as shellfish culture) could favor species transfers between those two provinces (Bachelet et al. 2004; Fernández-Rodríguez et al. 2022). Direct ship traffic between the naval bases of Toulon and Brest (Atlantic), where W. subatra is common (see Gauff et al. 2022; Rondeau et al. 2022) might further increase this risk of introduction. It is, therefore, not unexpected to find this species in a large Mediterranean harbor like Toulon.

More troubling is the high abundance of this species, suggesting that it is well established and has been present for some time already. This species seems to have been present for at least four years in the Mediterranean, as a previous study in Marseilles has tentatively identified the species in 2019 (Gauff et al. 2023b). Watersipora subatra seems to be the most dominant Watersipora species in the French Mediterranean as we did not detect any W. subtorquata despite this species being identified in the Toulon Darse Nord Marina in past studies Gauff et al. 2023a)). This might suggest that the species has been misidentified as W. subtorquata for several years. The comparatively low number of taxonomic experts still in full activity in this area might explain such misidentifications/absence of identifications (Ferrario et al. 2018). Taxonomic expertise requires much time and rigor (Caley et al. 2013; Coleman and Radulovici 2020). Currently, there is an increasing tendency to substitute taxonomy with time-efficient genetic tools to either confirm species identifications by barcoding (Liu et al. 2017; Kenworthy et al. 2018) or to detect species in an area via metabarcoding (Leray and Knowlton 2015; Miralles et al. 2016; Ardura and Planes 2017; Couton et al. 2019, 2022; Azevedo et al. 2020). These tools can be quite powerful to detect NIS, especially when combined with morphological analyses (Azevedo et al. 2020; Couton et al. 2022), however the efficiency of the method depends on the quality of reference sequences (Viard et al. 2019; Couton et al. 2022). Species identification based on molecular methods still require detailed morphological identifications to be carried out when reference sequences are generated. Integrative taxonomy, where genetics and morphology are both carefully considered, is required in order to ensure accurate species identifications (Dayrat 2005). Monitoring programs that use eDNA or other molecular approaches require reliable databases, with reference sequences generated from specimens that have been carefully identified or updated once errors are detected. The lack of genetic references for some species can result in missing or misidentified NIS and other species using metabarcoding (Couton et al. 2022). Even more problematic however, are reference sequences with misidentifications, as they provide a false sense of certitude to authors without taxonomic expertise (Viard et al. 2019; Cognato et al. 2020). Vieira et al. (2014) first noted that high numbers of Watersipora identifications (ex. Mackie et al. 2006; Mackie et al. 2012) were erroneous and our phylogeny has since revealed that 49% of Watersipora sequences on Genbank were incorrectly identified, a percentage that rises to 65% when excluding sequences identified only to the genus level. Most misidentifications concern W. subatra being listed as “W. subtorquata”. This causes subsequent identification errors in publications using these reference sequences (ex: Duncan et al. 2022; Miralles et al. 2016; Portas et al. 2022) This problem with non-updated sequences has already been pointed out in the past, as it prevents clear conclusions on species identity and origin (Miralles et al. 2018). This might explain why W. subatra has not been detected for a long time in the Mediterranean Sea. One must note that a recent checklist of NIS in France (Massé et al. 2023) includes W. subatra in the Atlantic and Mediterranean as an established species, but the absence of W. subtorquata in this list suggests that both species are potentially synonymized. Our updated genetic reference list could be used as a guide by authors having deposited Watersipora sp. sequences to update their species identity.

Our phylogeny includes two problematic species identifications: “Watersipora sp. sensu Mackie et al. (2012)” and “W. edmondsoni”. The first can be attributed to the genetic description of a novel species from Mackie et al. (2012). This species however lacks a morphological description and could thus be an already described species that simply lacks a corresponding genetic sequence for now. It may be Watersipora atrofusca, as it co-occurs with “Watersipora sp. sensu Mackie et al. (2012)” in California. However a record of “Watersipora sp. sensu Mackie et al. (2012)” identified as “W. subtorquata” in Korea (HQ896194, Lee et al. 2011) suggests one of three alternatives: either W. atrofusca is also present in Korea, another species corresponds to “Watersipora sp. sensu Mackie et al. (2012)”, or “Watersipora sp. sensu Mackie et al. (2012)” does indeed constitute an undescribed species. Without morphological identification of the individuals, inference on these three options remains speculative. “Watersipora edmondsoni” constitutes a similar problem. The holotype of W. edmondsoni Soule & Soule, 1968 was reexamined by Vieira et al. (2014) and has been reattributed as synonymous to W. subtorquata. We here however note a genetically distinct clade containing the individuals identified as “W. edmondsoni” by two separate studies (Mackie et al. 2006; Suppl. material 1: table S1 MW277712). Vieira et al. (2014) suggested that specimens reported as W. edmondsoni in Soule and Soule (1975) could indeed include one or more species. The identified sequences could thus potentially be attributed to W. edmondsoni sensu Soule and Soule (1975) (non Soule & Soule, 1968). Further specimens and sequences are required to resolve the species status of specimens reported as “W. edmondsoni”.

Due to the high damages of NIS on ecosystems and the economy (Blackburn et al. 2019; Diagne et al. 2021), they constitute a key global biosecurity issue (Lovell et al. 2006; Olson 2006; Pyšek et al. 2020). Proper monitoring of their spread is thus crucial for preventing or mitigating their impact (Pyšek et al. 2020). This however requires correct identification, as the species identity may impact how we evaluate NIS invasion patterns (Vieira et al. 2014; Viard et al. 2019; Golo et al. 2023). Here we show that some authors may have been misled due to misidentifications of Watersipora spp. (prior to its’ redescription) in the reference literature and in genetic reference banks. We suggest that authors should maintain genetic references in accordance with new research by including the name originally used on their research and potential changes to their ID after reexamination. However, this would be very time consuming. A new way of genetic database management, similar to the WoMRS database, might compensate for the time-consuming nature of follow-up corrections on sequences. Taxonomists and geneticists dedicated to a family/genus could have a right to modify scientific names associated with sequences following the recommendations of recent peer-reviewed papers. Changes (by whom, references, etc.) should be tracked for transparency. This could help avoid the perpetuation of errors and improve the monitoring of both the spread of NIS and species distributions in general. Our new sequences, as well as the table updating the identity of almost all existing Watersipora sequences (Suppl. material 1: table S1) may help with future identification of different Watersipora species.

Funding declaration

This study was supported by the Région Bretagne through the CoEcoDigue project. (Ref/Région n° 2017-01, Ref/Ifremer n°21/1001756) and by the Chambre de Commerce et d’Industrie (CCI) du Var. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors’ contribution

Robin P. M. Gauff: research conceptualization, methodology, investigation and data collection, data analysis and interpretation, writing – original draft; Marc Bouchoucha: research conceptualization, investigation and data collection, funding provision, writing – review & editing; Amelia Curd: research conceptualization, funding provision, writing – review & editing; Gabin Droual: research conceptualization, investigation and data collection, data analysis and interpretation, writing – review & editing; Justine Evrard: investigation and data collection, data analysis and interpretation, writing – review & editing; Nicolas Gayet: investigation and data collection; Flavia Nunes: research conceptualization, methodology, investigation and data collection, data analysis and interpretation, funding provision, writing – review & editing.

Acknowledgements

We wish to thank the harbor authorities of the Toulon Darse Nord marina, La Seyne sur Mer marina and Old Harbor of Marseilles for authorizing access for sampling of Watersipora colonies. We also wish to thank Jasmine Ferrario (University of Pavia) and Leandro Manzoni Vieira (University of São Paulo) for discussions on Watersipora spp. We wish to also thank the reviewers of this article.

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Supplementary materials

Supplementary material 1 

Watersipora sequences retreived from Genbank

Robin P. M. Gauff, Marc Bouchoucha, Amelia Curd, Gabin Droual, Justine Evrard, Nicolas Gayet, Flavia Nunes

Data type: table (Excel spreadsheet)

Explanation note: table S1: Watersipora sequences retreived from Genbank with their original identification, location, and reference as well as their new species attribution according to our phyllogenetic tree (See Suppl. material 3: fig. S1).

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (851.60 kb)
Supplementary material 2 

Geographic references for the distribution of various Watersipora spp.

Robin P. M. Gauff, Marc Bouchoucha, Amelia Curd, Gabin Droual, Justine Evrard, Nicolas Gayet, Flavia Nunes

Data type: table (Excel spreadsheet)

Explanation note: table S2: Geographic references for the distribution of various Watersipora spp. used for generating Fig. 4 (note that species identity here corresponds to our identification and not necessarily to the original one).

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (13.45 kb)
Supplementary material 3 

Combined Bayesian (BEAST) and Maximum likelihood (iqtree) phylogenetic tree

Robin P. M. Gauff, Marc Bouchoucha, Amelia Curd, Gabin Droual, Justine Evrard, Nicolas Gayet, Flavia Nunes

Data type: figure (JPG file)

Explanation note: figure S1: Combined Bayesian (BEAST) and Maximum likelihood (iqtree) phylogenetic tree of all Watersipora COI sequences available on Genbank (With accession number and corresponding source references). Numbers at the nodes correspond to the posterior probabilities from the ‘BEAST’ analysis followed by the ultra-fast bootstrap values from ‘iqtree’, both expressed as percentages (i.e., 100/100). All accession number were listed in this extended version compared to Fig. 3. New sequences acquired in this study are indicated by *. Color coding refers to species names listed on Genbank. Corrected identification (See Suppl. material 1: table S1) are: Clade 1 W. arcuata; Clade 2 W. subtorquata; Clade 3 “Watersipora sp. sensu Mackie et al., (2012)”; Clade 4 “W. edmondsoni”; Clade 5 W. subatra.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (1.33 MB)
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