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Mediterranean spreading of the bicolor purse oyster, Isognomon bicolor , and the chicken trig-ger, Malleus vs. the Lessepsian prejudice

The introduction rate of alien species in the Mediterranean Sea is rapidly growing, and their taxonomical identification is increasingly challenging. This uncertain identification often leads to an incorrect estimation of the number of alien species, their route of introduction, and their potential negative effects. This is particularly true for some bivalves, which are characterized by high variation in their shells, resulting in uncertain morphological identification. This is the case for two alien bivalves, i.e., an Isognomonidae and a Malleidae species, both characterized by confused historical colonization records in the Mediterranean Sea, misidentifications, and controversial and changing nomenclatures that have insofar negatively affected our knowledge on their geographical distributions. In this respect, molecular approaches provide a strategy that is especially useful when traditional taxonomy fails, and DNA barcoding is a powerful and well-known tool to obtain reliable identifications through efficient molecular markers. In this work, we used the 16S rRNA marker to assess the preliminary identification of Isognomon sp. and Malleus sp. specimens from different localities in the Southern Mediterranean Sea. Bayesian inference (BI) and maximum likelihood (ML) methods were applied to test the monophyly of the phylogenetic linages and to clarify their taxonomic positions, allowing a com-plete overview of the colonization and spreading of these two alien bivalves in the Mediterranean Sea. In particular, the Isognomon sp. specimens were identified as the Atlantic I. bicolor , highlighting that previously suggested invasive migration patterns, (i.e., the Lessepsian migration), must be reconsidered with stronger critical attention in light of currently occurring global changes.


Introduction
The first record of the non-indigenous genus Isognomon [Lightfoot], 1786 in the Mediterranean Sea is due to Mienis (2004), who reported on an unspecified number of Isognomon ephippium (Linnaeus, 1758) specimens collected in Israel. Later, Zenetos et al. (2005) and Galil (2007) questioned the occurrence of this species, as it was recorded only once on an oil platform from the Red Sea. Still in Israel, a single initially undetermined "Hammer or Tree oyster" shell, was found in 2015 and, based on the iconography of Oliver (1992: 67, plt 10, figs. 4a-f) and Huber (2010: 176, text-figure), was identified by Mienis et al. (2016) as Isognomon legumen (Gmelin, 1791). Afterwards, the settlement of this latter species along the Israelian coast was confirmed by Marchini et al. (2020) and reported from Turkey by Ovalis & Zenetos in Stamouli et al. (2017) and from Greece by Angelidis in Lipej et al. (2017). The latter group mentioned the previous misidentification of I. legumen with the co-occurrent Malleus regula (Forsskål in Niebuhr, 1775) (see Angelidis in Lipej, 2017) and traced the establishment of this alien bivalve in the Aegean Sea to 2016. However, specimens reported as M. regula from Lybia were subsequently re-identified as I. legumen by Crocetta (2018), who suggested that this species might have occurred in the Mediterranean Sea before 1996. Concurrently, its rapid spread westward in the Mediterranean basin was confirmed by reports from the Ionian coast of Sicily (Scuderi & Viola, 2019) and the Strait of Messina (Giacobbe & Renda in Dragičević et al., 2019), whilst Angelidis & Polyzoulis (2018) reported I. legumen and I. australica (Reeve, 1858) (first record from the Mediterranean Sea) as sympatric in the South Aegean. The occurrence of I. australicus in the Eastern Mediterranean was confirmed by Manousis (2021). In this regard, we noted that juveniles I. legumen reported from Greece by Micali et al. (2017), and other specimens reported as "Malleus regula" by Angelidis in Lipej et al. (2017) but re-identified as I. legumen by Crocetta et al. (2017), appear very similar to the I. australica specimens outlined by Angelidis & Polyzoulis (2018). However, according to Zenetos et al. (2018) "the contemporary presence in Astypalaia of two taxa belonging to Isognomon Lightfoot, 1786, one of which apparently absent from the Red Sea, seems unlikely", so that the same researchers preferred to ascribe both records to the Red Sea taxon I. legumen "to avoid the inflation of alien molluscan records and pending molecular confirmations". Partially in agreement with this latter opinion, Albano et al. (2021) prudentially reported "I. aff. australica (sensu Angelidis and Polyzoulis, 2018)" from Cyprus and Kriti. The current literature thus demonstrates how our knowledge on the genus Isognomon in the Mediterranean Sea is complicated by a series of misidentifications, conflicting opinions and interpretations. This is unsurprising when we consider that the genus Isognomon is characterized by marked habitat-dependent variability in shell shapes (Fischer-Piette 1976;Coan et al., 2000;Wilk & Bieler, 2009) and convergent interspecific morphologies (Benthotage et al., 2020). These features make shell-based discrimination in the species unreliable, as shown by Wilk & Bieler (2009) for I. alatus (Gmelin, 1791) vs. the sympatric I. bicolor (C.B. Adams, 1845) and the allopatric I. ephippium in the Florida Keys. Morphological similarities and shell shape variability may also explain the presumptive record from the Canary Islands of the exclusively Indo-Pacific I. isognomum (Linnaeus, 1758) (Gömez Rodriguez & Perez Sanchez, 1998), which should instead be assigned to the West African I. dunkeri (P. Fischer 1881), according to Huber (2015), or to the West Atlantic I. bicolor, according to Holmes et al. (2015). The entire systematics of the genus Isognomon, however, is controversial and requires an update supported by molecular data. For example, according to Huber (2015) the phylogenetic data of Tëmkin (2010) indicates that the two most common Indo-Pacific Isognomonidae, i.e., Isognomon (I.) ephippium (Linnaeus 1758) and I. (I.) isognomum (Linnaeus 1758), may be derived from two sibling species. It follows that a resolution of valid names, with the related species distributions and habitat preferences, requires thorough review, necessarily supported by detailed illustrations of the genetically analysed specimens. Similar considerations may concern the above-mentioned M. regula, whose putative spread across the Mediterranean remains uncertain, also due to the frequent erroneous attribution of Isognomon sp. specimens (Crocetta et al., 2017;Crocetta, 2018). Malleus regula, although a precocious Lessepsian migrant [the first Mediterranean record from Syria and Lebanon by Moazzo (1931)] has been reported as established in the Levantine Basin only within the last decade (Marchini et al., 2020) and in the Ionian Sea even more recently (Prato & Rubino in Kousteni et al., 2019;Bini & Pina-sco, 2021), with an apparent disjunct distribution which could indicate unequal investigation efforts. In this paper, the first molecular identification of the above-mentioned presumptive species is provided through DNA barcoding and a phylogenetic approach using Bayesian inference (BI) and maximum likelihood (ML) methods. We use these data to clarify the taxonomic position of two controversial taxa, providing insight on the patterns and processes of their introduction and spread in the Mediterranean Sea.

Monitoring and sampling
Monitoring of Non-Indigenous Species, NIS, starting in the framework of the PRA 2008/2009 program "Settlement dynamics and colonization of allochthonous assemblages in the Capo Peloro Lagoon" (Strait of Messina, central Mediterranean), was gradually extended to the adjacent Tyrrhenian and Ionian basins and integrated with further investigation in North Ionian, Aegean, and Levantine Sea (PhD school contribution 2016; PON 2014-2020, grant AIM 1848751-2). Two sampling methods have been employed, i.e., by hand collection during snorkelling and SCUBA recognition and scraping of 30x30 cm hard bottom surface. The scraped samples, retained in a fine net (< 100 µm), were fixed in ethanol 70% in the field or, whenever possible, rapidly brought in the laboratory and immediately frozen. Subsequently, an accurate sorting was carried out under a stereomicroscope, and the selected specimens definitively fixed in 70% or 95% ethanol, depending on their destination for analysis. Shell characters are investigated to propose a morphological identification comparing the shells of the new Mediterranean specimens with shells of previous works (Huber, 2010;Mienis et al., 2016;Micali et al., 2017;Ovalis & Zenetos in Stamouli et al., 2017;Angelidis & Polyzoulis, 2018;Crocetta, 2018;Giacobbe & Renda in Dragičević et al., 2019;Prato & Rubino in Kousteni et al., 2019;Scuderi & Viola, 2019;Marchini et al., 2020;Albano et al., 2021;Bini & Pinasco, 2021;Manousis et al., 2021), and pictures of shell specimens of I. bicolor (AMNH:307896) from Florida Keys (USA) and M. regula (AMNH:319335 and AMNH:319339) from Darwin Harbour, Australia stored at the American Museum of Natural History (AMNH).

Molecular analysis
A piece of adductor muscle was clipped from each sample and total DNA was extracted using a standard high-salt protocol (Sambrook, 1989). Amplification of the 16S fragment gene was performed by PCR using universal primers 16Sar-L and 16Sbr-H (Palumbi et al., 1991). The PCR conditions used were: 3 min denaturation step at 94°C; 35 cycles of 94°C/60 s, 51°C/60 s, 72°C/60 s; 10 min final extension at 72°C. Amplicons were sequenced by Genewiz GmbH (Germany) (https:// www.genewiz.com). Sequences are deposited in Gen-Bank database (https://www.ncbi.nlm.nih.gov/Gen-Bank/) with accession numbers OK104096-OK104098 and OK166813. The Basic Local Alignment Search Tool (BLASTN) search (Altschul et al., 1990, Johnson et al., 2008 was used in a preliminary identification of the sequences, and also to exclude contaminations. In addition, two 16S datasets, one for each family (Isognomonindae and Malleidae), were built including the newly produced sequences and all the GenBank sequences belonging to each of the families. Datasets were aligned with MAFFT v.7 online service (Katoh et al., 2019) using the E-INS-i iterative refinement algorithm. Polymorphic sites of the three dataset alignments were calculated using DnaSP 6 (Rozas et al., 2017). Uncorrected p-distances and Kimura 2-paramer (K2p) distances were calculated using the program Mega X (Kumar et al., 2018). A third dataset, used for downstream phylogenetic analysis, was built by joining together the two previous datasets, and included the addition of Atrina rigida ([Lightfoot], 1786) (HQ329397) as an outgroup. The GTR+G model was selected as the best evolutionary model by JModelTest 0.1 (Posada, 2008) according to the Bayesian information criterion (BIC). Phylogenetic analyses were performed using Bayesian inference (BI) and maximum likelihood (ML) methods. BI was carried out using MrBayes 3.2.7 (Ronquist et al., 2012) running two Markov chains of 5 000 000 generations each, sampled every 1000 generations. Consensus trees were calculated on trees sampled after a burn-in of 25%. The ML tree was inferred with W-IQ-TREE web platform (Trifinopoulos et al., 2016) using an ultrafast bootstrap (uBS) analysis with 1000 generations. The obtained phylogenetic trees were visualised and curated using FigTree v.1.4.4 (available at http:// http://tree. bio.ed.ac.uk/software/figtree/).
A juvenile Malleus sp. (maximum size 12 mm) was collected at Polis, Cyprus (35.043453°N, 32.416217°E) Levantine Basin, in June 2019, on a rocky bottom substrate at 2 m depth, as an epibiont on Chama pacifica Broderip, 1835 and was analysed using molecular methods (Table 1). Additional specimens were sampled in July 2020, in "Mar Piccolo di Taranto" (40.481283° N, 17.268383° E), a sheltered area of the Ionian Apulian coast (Italy) (Fig. 1). The Malleus sp. specimens were found as epibionts on rocks (Fig. 4) and anthropic structures at 3 m depth, or as dead shells (maximum sizes ranging from 12 mm to 46 mm; Fig. 5).

Molecular analyses
It was impossible to unequivocally assign morphological identifications for any new Mediterranean specimens. Therefore, the Mediterranean samples, Isognomon sp. specimens from Briatico (OK104096) and from Messina (OK104097) and Malleus sp. specimens from Cyprus (OK104098) and from Taranto (OK166813) were analysed using the 16S mitochondrial barcode marker. BLASTN results of OK104096 and OK104097 nucleotide sequences showed 100% of similarity with the GenBank sequence identified as I. bicolor (HQ329406; voucher AMNH:307896). BLASTN results of OK104098 and OK166813 sequences showed less than 90% of similarity with any sequences stored in GenBank as Malleus spp. (Table 1). Therefore, it was only possible to assign an identification at genus level.
In addition, we built three different 16S sequence datasets. The 16S Isognomonidae sequences alignment consisted of 503 positions and a total number of 124 polymorphic sites. The 16S Malleidae sequences alignment       Table 2). Results of the distance analyses showed no intraspecific divergence between each couple of new sequences (OK104096/OK104097 and OK104098/OK166813). Moreover, the interspecific genetic distance (p-distance/K2p) in Isognomonidae family ranged from 4.6%/4.8% to 23.1%/29%, while in Malleidae the genetic distances ranged from 5.1%/5.3% to 15.9%/18.1%. The phylogenetic results corroborate our preliminary identification attempts (Fig. 6). Bayesian posterior probabilities (BPP) and ultrafast bootstrap (uBS) values, based respectively on BI and ML analyses, are reported at each node of the tree. Each of the new sequenced specimens were placed in two distinct highly supported clades: BPP=1; uBS ≥98%.

Taxonomic investigation of Mediterranean Isognomon sp. and Malleus sp. specimens
The molecular data generated here provides compelling evidence that our Mediterranean Isognomon sp.   Briski et al., 2016). However, our 16S sequences (OK104096 and OK104097) showed a 100% matchrate with a reference sequence from museum specimen (AMNH:307896) sampled in the Florida Keys (present- ed in Fig. 2A-D). This sampling locality is north of the Caribbean region where the type locality of I. bicolor is found (Jamaican coasts) (Adams, 1845). In addition, this determination agrees with the redescription of I. bicolor by Domaneschi & Martins (2002), although this similarity cannot be considered by itself definitive proof, due to the well-known morphological variability characterizing Isognomon species (Wilk & Bieler, 2009). In this regard, the small number of ligamental resilifer teeth observed in the specimens that were analysed using molecular tools ( Fig. 2I-J) can be explained by the high variability in this structure within the Isognomon genus, which in fact does not constitute a diagnostic character (Domaneschi & Martins, 2002). The 16S sequences of Malleus sp.1 (OK104098 and OK166813) did not match any sequences present in GenBank, leaving such specimens undetermined at the species level. In particular, Malleus sp.1 sequences are 10.4% distant from M. regula sequences of museum specimens (AMNH:319335 and AMNH:319339 from Australia; Fig. 5A-F). Moreover, Malleus sp.1 and Malleus regula sequences formed two well-separated and highly supported clades in the Bayesian phylogenetic tree (Fig. 6). Molecular identification of previously misidentified alien Isognomon sp. And Malleus sp. Specimens from the Mediterranean Sea (Tyrrhenian, Ionian, and Aegean basins) have been provided here for the first time using the 16S rRNA marker. Furthermore, phylogenetic results based on BI and ML analyses confirmed that the analysed Mediterranean specimens belong to Isognomonidae and Malleidae families, representing two distinct well-supported clades.

Atlantic introduction
The molecular identification of I. bicolor brings into question all previous morphological identifications of Isognomonidae in the Mediterranean, whose shell morphologies always agree with the variability range of I. bicolor. It is remarkable that observations on the I. bicolor invasiveness and habitat preferences in the west Atlantic also agree with reports from the Mediterranean. As invasive species, for example, the "bicolor purse oyster", originating from the native Caribbean region (Benthotage et al., 2020), has spread southward, reaching the north-eastern Brazilian coasts in the 1970s (Loebmann et al., 2010;Dias et al., 2013;De Oliveira Gomes & Carvalho Da Silva, 2013), and then rafting to the Uruguayan coast (Breves et al., 2014), representing one of the most Fig. 6: Bayesian phylogenetic tree based on the joined 16S dataset. Bayesian posterior probability (left) and ultrafast bootstrap (right) values are indicated at each node. *: indicates the specimens sequenced in this work. Sequences of AMNH museum specimens of Isognomon bicolor (Fig. 2) and Malleus regula (Fig. 5) used for morphological comparisons are respectively HQ329406, and HQ329413/HQ329414. important introductions to the benthic realm (Ferreira et al., 2009). As for the Mediterranean reports, in Brazil this species was initially misidentified, i.e., with the alleged endemic I. alatus (Gmelin, 1791) (Domaneschi & Martins, 2002). In south Brazil, moderate wave exposure and availability of heterogenous steep rocky substrates (Whorff et al., 1995;Domaneschi & Martins, 2002) and rock crevices (Moyses, 2005;Zamprogno et al., 2010), seem to be important for the invasive success of I. bicolor, which preferentially settles inside biogenic secondary substrates (Rocha, 2002). Such observations, as well as suggested associations in invaded environments with native macroalgae (López & Coutinho, 2010) and vermetid reef habitat (Breves & Junqueira, 2017), substantially agrees with our sampling data. This habitat preference suggests that I. bicolor, despite the high invasiveness, cannot be considered a pioneering species in substratum colonization (Rocha, 2002) and does not display a clear opportunistic behaviour (Benthotage et al., 2020). Ballast water and/or fouling of international commercial vessels (Breves-Ramos et al., 2010;Fernandes et al., 2016) and relocation of oil or gas platforms from the Caribbean coast (Oliveira & Creed, 2008;Breves-Ramos et al., 2010) have been considered possible vectors of I. bicolor to Brazilian waters, although others have reported on the role of rafting by floating debris (Breves et al., 2014). This latter suggestion agrees with the known use of floating macroalgae by I. bicolor as a secondary dispersal pathway (Dias et al., 2013) and also explains its stranding in south England as an epibiont on a plastic spool, almost simultaneously with other Caribbean molluscan species (Holmes et al., 2015). Such mechanisms for west to east Atlantic dispersal of southern, warm water taxa (Holmes et al., 2015), cannot be separated from the Mediterranean spreading of tropical neuston communities, which occasionally even reach the cold Liguro-Provençal basin (Betti et al., 2017). Moreover, the northward shift of the 15°C February isotherm divide, providing a warmer corridor which directly connects the Strait of Gibraltar with the eastern Mediterranean (Bianchi et al., 2012), allows tropical taxa to bypass most of the western Mediterranean Sea (Di Silvestro et al., 2010), rapidly reaching the most favourable Levantine Basin throughout the Mid-Mediterranean Jet Current. Genetic investigations have evidenced that these patterns of invasion have occurred frequently in other taxa; for example, the warm Atlantic Heterobranchia Aplysia dactylomela Rang, 1828, settled in the eastern Mediterranean before spreading westward and northward (Valdés et al., 2013). Similarly, secondary dispersal from Aegean might be responsible for the Ionian colonization by I. bicolor which, according to our data, initially settled in the eastern coasts of Sicily (Scuderi & Viola, 2019, as I. legumen), and subsequently spread northward throughout the Strait of Messina, rapidly colonizing the Southern Tyrrhenian coasts, according to the local sea-surface circulation (Vetrano et al., 2010). We thus agree with Por (2009), who considered the attention towards the Lessepsian migrants disproportionately greater than the increasing settlement by tropical Atlantic newcomers through the Straits of Gibraltar. Among oth-ers, a consequence of this is that when non-native species belonging to tropical genera or families first appear in the Eastern Mediterranean they may be classified according to the most similar Indo-Pacific taxon, implicitly excluding other possibilities. Such Lessepsian prejudice, allowing a cascade of misidentifications, could preclude early warning of potential threatening invaders, such as I. bicolor. This invasive species, which in Brazilian coasts has caused profound changes in the native rocky coast communities reaching densities up to 800 individuals/100 cm 2 (Magalhães, 1999;Rapagnã, 2004;Breves-Ramos et al., 2010), was locally responsible for reducing 50% of the native barnacle population, Tetraclita stalactifera Lamarck, 1818 (López, 2003), and probably competes with Perna perna (Linnaeus 1758), an edible mussel of commercial interest (Rapagnã 2004;Breves-Ramos et al., 2010). In this respect, the observation that this invasive species does not settle on bare substrate, appearing only at late succession stages (Rocha, 2002), is not reassuring, and thereby represents a potential threat for protected species and habitats, such as the Mediterranean vermetid reefs (Milazzo et al., 2016), whose analogues in Brazilian waters are competing with I. bicolor colonization (Breves & Junqueira, 2017).
The spreading of Malleus sp.1 in the Mediterranean, confirmed by a sample from Cyprus, strongly questions previous records regarding the establishment of M. regula in Greek and Turkish Aegean waters (Giannuzzi-Savelli et al., 2001;Crocetta et al., 2017), in the Ionian Sea (Prato & Rubino in Kousteni et al., 2019), as well as recent records from Sicily (Bini & Pinasco, 2021). In the meantime, these observations cast doubts about both historical and recent reports from the Levantine Basin. Whilst we wait to understand the true identity of this undetermined taxon through molecular and morphological screening of the entire genus, its spread needs to be carefully monitored, since it represents a potential threat for biodiversity conservation in the Mediterranean.

Conclusions
Increasing anthropogenic introductions and climate change have expanded the frequency of alien invasions and the variety of ecoregion sources of potential invaders. Invasive patterns, which were previously taken for granted, such as the Lessepsian migration, must be reconsidered with stronger critical attention. This changing scenario highlights an even stronger need for accurate taxonomic identification, from which more reliable predictions of settlement patterns and potential ecosystem damage can be derived. Despite the occurrence of an increasing number of marine alien species in the Mediterranean Sea, taxonomical identification of these invasive taxa is often still difficult when based on morphological characters alone. This uncertain identification often leads to an incorrect estimation of the number of alien species and their true routes of introduction. This is particularly true for bivalves characterized by a high variation in their shells, leading to uncertainty in morphological identifica-tion. This is the cases for two alien bivalves assigned to Isognomonidae and Malleidae families characterized by their confused Sea of origin and their controversial and changing nomenclature. The variable phenotype of their shells has historically produced several cases of misidentification, which has negatively affected our knowledge on their geographical distribution to date. In this respect, molecular approaches using DNA barcoding and phylogenetic analyses are fundamental tools to avoid the misidentification and related hasty conclusions, mainly when traditional taxonomy fails. In this work, the 16S rRNA marker was utilized for the first time to assess the identification of Isognomon sp. and Malleus sp. specimens from different localities of the southern Mediterranean Sea. The first taxon is now assigned as the Atlantic species I. bicolor, whilst the unassigned second taxon should be referred as Malleus sp.1 instead of the Indo-Pacific M. regula. Obviously, indispensable tools such as GenBank are still far from offering the full coverage required for our current needs, which is why a greater effort should be devoted to integrating known families or genera suspected to be more involved in invasive processes.