High genetic diversity and lack of structure underlie the invasion history of the non-indigenous oyster Dendostrea cf. crenulifera (Mollusca, Ostreida, Ostreidae) spreading in the eastern Mediterranean Sea

277 High genetic diversity and lack of structure underlie the invasion history of the non-indigenous oyster Dendostrea cf. crenulifera (Mollusca, Ostreida, Ostreidae) spreading in the eastern Mediterranean Sea Nathan Delcour1,2* , Matteo Garzia1* , P. Graham Oliver3, Emanuele Berrilli1, Andrea Toso4,5 , Michel Bariche6, Paolo G. Albano7, Paolo Mariottini8, Daniele Salvi1 1 Department of Health, Life & Environmental Sciences, University of L’Aquila, Via Vetoio snc, 67100 L’Aquila-Coppito, Italy 2 Faculty of Science and Technology, University of Lille, 42 rue Paul Duez, 59000 Lille, France 3 National Museum of Wales, Cathays Park, CF10 3NP Cardiff, Wales, UK 4 Department of Biological and Environmental Sciences and Technologies - DiSTeBA, University of Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy 5 National Biodiversity Future Center (NBFC), 90133 Palermo, Italy 6 Biology Department, American University of Beirut, Beirut 1107 2020, Lebanon 7 Department of Marine Animal Conservation and Public Engagement, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy 8 Department of Science, University of “Roma Tre”, Viale Marconi 446, 00146 Rome, Italy Corresponding author: Daniele Salvi ([email protected]) Copyright: © Nathan Delcour et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Since the opening of the Suez Canal in 1869, hundreds of Indo-Pacific species have rapidly colonised the Mediterranean. Understanding the spatial and temporal patterns of this biological invasion is crucial for assessing its ecological impact. A notable example is the non-indigenous oyster Dendostrea sp., first discovered in Türkiye in 1998 and later found throughout the easternmost Mediterranean, though its identity remained uncertain. This study clarifies the taxonomic identity and the introduction pathways of Dendostrea sp. using molecular analyses. Over 100 specimens from 25 sites in the eastern Mediterranean, as well as Île d’Ambre and Rodrigues in the Mauritius Archipelago (the native range), were sequenced for mitochondrial DNA (COI) and compared to 422 sequences from GenBank. Phylogenetic and species delimitation analyses identified the Mediterranean oysters as D. cf. crenulifera, conspecific with oysters from Rodrigues. The Mediterranean populations exhibited high genetic diversity, lack of phylogeographic structure and showed no evidence of a founder effect. These findings suggest that D. cf. crenulifera entered the Mediterranean over two decades ago through multiple shipping-mediated introductions from its native range and successfully established, likely aided by the decline of native biodiversity. The observed genetic diversity pattern across the Mediterranean indicates high propagule pressure driving the species’ invasion history, which likely underpins its establishment success by reducing the deleterious consequences of population bottlenecks and overcoming the so-called genetic paradox. This study underscores the value of molecular surveys in identifying taxonomically challenging non-indigenous species and uncovering their invasion histories. Key words: Alien, bivalve, cox1, phylogenetics, phylogeography, systematics, true oyster Academic editor: Marcela Uliano-Silva Received: 7 April 2025 Accepted: 7 August 2025 Published: 3 October 2025 Citation: Delcour N, Garzia M, Oliver PG, Berrilli E, Toso A, Bariche M, Albano PG, Mariottini P, Salvi D (2025) High genetic diversity and lack of structure underlie the invasion history of the non-indigenous oyster Dendostrea cf. crenulifera (Mollusca, Ostreida, Ostreidae) spreading in the eastern Mediterranean Sea. NeoBiota 101: 277–302. https://doi. org/10.3897/neobiota.101.154917 NeoBiota 101: 277–302 (2025) DOI: 10.3897/neobiota.101.154917 * These authors contributed equally to this work. Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 278 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Introduction Increased international trade and transportation, combined with ongoing global warming, have accelerated global biotic homogenisation (Occhipinti-Ambrogi and Galil 2010). The shifts in environmental conditions and the weakening of ecological and geographical barriers have facilitated the introduction and establishment of numerous marine non-indigenous species (NIS) outside their native ranges, reaching even the most remote places on the Planet (Occhipinti-Ambrogi and Galil 2010; Ardura et al. 2021). Some NIS are fast expanding species that quickly establish in local communities leading to sudden changes and increasing pressures on native species (i.e. by predation, changes in habitats through ecosystem engineering and the introduction of pathogens and parasites). NIS introductions also affect various socio-economic services, including fisheries and aquaculture, human health and infrastructure (Streftaris and Zenetos 2006; Tsirintanis et al. 2022). The Mediterranean Sea, particularly its eastern sector, is currently experiencing a collapse of native biodiversity and is home to a massive invasion of tropical species entering from the Suez Canal or transported by shipping (Albano et al. 2021a, b; Toso et al. 2024). In 2021, almost 1000 NIS were reported in the Mediterranean Sea, with established species increasing by 40% since 2010 (Zenetos et al. 2022). Indeed, establishment rates also increased in the last decade, from ~ 3% yearly during 2011–2021 to ~ 6% between January 2020 and December 2021 (Zenetos et al. 2022). Mollusca are amongst the most diverse marine NIS, with 230 taxa representing more than 20% of the NIS established in the Mediterranean Sea (Zenetos et al. 2022). The vast introduction of molluscan NIS into the Mediterranean Sea is primarily attributed to international maritime traffic (specifically through ballast water and hull fouling) and the opening of the Suez Canal that broke a long-standing biogeographic barrier (Galil 2008; Crocetta et al. 2013; Nunes et al. 2014; Tempesti et al. 2020). One of the main dispersal pathways into the Mediterranean is thus from the Red Sea and the broader Indo-West Pacific realm, with the eastern Mediterranean being particularly impacted due to its proximity to the Canal (Galil 2008; Zenetos et al. 2009; Nunes et al. 2014; Galil et al. 2015a, b; Tempesti et al. 2020). This process, known as Lessepsian invasion (Por 1978), has been enhanced by the widening of the Suez Canal in 2015 (Galil et al. 2015a, b) that allowed greater hydraulic, hence biological, connectivity and ship traffic. Additionally, the global increase in sea surface temperatures (SST) and salinity in the eastern Mediterranean Sea and the subsequent local extinctions of native species that make resources available (Albano et al. 2021a) are facilitating new introductions and the intra-basin spread of those that have occurred already (Raitsos et al. 2010; Albano et al. 2021a). The presence of ecotypes, species complexes and cryptic diversity amongst molluscs complicates the taxonomic identification of NIS and consequently the understanding of invasion patterns, geographic distribution and the number of taxa involved. Oysters (Bivalvia: Ostreidae, Rafinesque, 1815) are a particularly difficult case where misleading morphology and controversial or confused taxonomy hinder accurate species identification. This group of bivalves is understudied and morphological identification is challenging due to phenotypic plasticity and a lack of reliable morphological diagnostic characters (Lam and Morton 2006; Xia et al. 2009; Salvi et al. 2014; Guo et al. 2018; Salvi and Mariottini 2021). Molecular approaches have proven extremely useful in resolving taxonomic uncertainties and 279 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera ensuring accurate species identifications (Wang et al. 2004; Raith et al. 2015; Pejovic et al. 2016; Cavaleiro et al. 2019; Al-Kandari et al. 2021; Salvi et al. 2021, 2022; McDougall et al. 2024). When combined with comprehensive population sampling, molecular approaches can provide insights into the genetic structure of NIS populations, crucial for understanding the invasion process and its success (Lee 2002; Wellband et al. 2017; Geburzi et al. 2020; Oyarzún et al. 2024). The difficulty in morphologically identifying oysters is well illustrated by the NIS of the genus Dendostrea Swainson, 1835, which is currently spreading throughout the eastern Mediterranean Sea. First detected in southern Türkiye in 1998 (Çeviker 2001), this species was subsequently recorded in Cyprus in 2008 (Zenetos et al. 2009), Greece in 2010 (Zenetos et al. 2011), Albania and Malta in 2016 (Ulman et al. 2017; Gerovasileiou et al. 2017) and Syria in 2019 (Ragkousis et al. 2023), with additional records reported in Cyprus, Greece, Türkiye and Syria between 2015 and 2021 (Karachle et al. 2016; Ulman et al. 2017; Ragkousis et al. 2023). Most authors attributed these records to the species Dendostrea frons (Linnaeus, 1758) (Çeviker 2001; Zenetos et al. 2009, 2011) based on shell features. The first molecular assessment of Mediterranean specimens of this non-indigenous oyster, using a DNA-barcoding approach, concluded that populations from the Aegean Sea (Greece) were conspecific with populations from the Levantine Sea (Türkiye), despite showing high phenotypic plasticity (Crocetta et al. 2015). However, the taxonomic identification was inconclusive because the closest match with GenBank sequences of Dendostrea folium (Linnaeus, 1758) had a genetic distance typically observed between distinct oyster species (Crocetta et al. 2015). In this study, we generate DNA sequence data from Dendostrea non-indigenous oysters collected through extensive sampling in the eastern Mediterranean Sea, as well as from its putative native range (western Indian Ocean) and compared with available DNA sequences and reference Dendostrea specimens in museum collections. The first aim of this study was to provide a molecular and taxonomic assessment of the Dendostrea oyster, with an improved sequence dataset including oysters from the Indian Ocean. The second aim was to perform population genetics analyses using an improved Mediterranean coverage of the species to identify potential pathways, vectors and processes driving this introduction and spread. Material and methods Sampling, DNA purification, amplification and sequencing Eighty oyster specimens, morphologically identified as putative Dendostrea sp., were collected from 25 sampling sites across the eastern Mediterranean. Sixteen specimens, whose dry shells are housed in the National Museum of Wales (NMW; Suppl. material 1), were collected from three sites in the Mauritius Archipelago, specifically two sites at Rodrigues Island and one at Île d’Ambre to fill the lack of GenBank sequences regarding the Indo-Pacific Ocean as stated by Crocetta et al. (2015). The dry shells of the Mediterranean specimens are stored in the Malacological Collection at the University of L’Aquila, the Stazione Zoologica Anton Dohrn and the Natural History Museum in Vienna as reference vouchers (Suppl. material 1). Specimens and coordinates of the sampling sites are reported in Table 1 and Suppl. material 1. The adductor muscle of each specimen was excised and fixed in 95% ethanol for molecular analysis. 280 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera DNA extraction was performed using the high-saline method (Sambrook 1989). The mitochondrial marker COI was amplified through PCR using primers LCO1490 and HCO2198 (Folmer et al. 1994), following protocols described in previous studies (Salvi et al. 2010; Crocetta et al. 2015). DNA presence was tested on 2% agarose gel electrophoresis. Sanger sequencing of PCR products was carried out by the company Genewiz® (www.genewiz.com), using the same primers employed for amplifications. Raw sequences were trimmed on Geneious Prime® v.2024.0.4 (Biomatters Ltd), using as a reference the complete mitochondrial genome of Crassostrea virginica (GenBank accession number AY905542; Milbury and Gaffney (2005)). Our dataset includes 96 specimens spanning eleven localities in Rhodes (Greece), six in Cyprus, six in Israel, one in Lebanon, two in Rodrigues and one in Île d’Ambre (Table 1, Fig. 1). Datasets and molecular analyses To perform a comprehensive phylogenetic assignment of the Mediterranean Dendostrea, a total of 422 COI sequences of Ostreinae species were mined from GenBank. These GenBank sequences and the 96 newly-generated sequences were combined in Dataset 1 (DS1), totalling 518 sequences. The DS1 was aligned using G-INS-i algorithm in MAFFT v.7.490 (Katoh 2002; Katoh and Standley 2013) under default parameters. A Neighbour-Joining (NJ) phylogenetic tree was built, based on genetic distances computed under the Kimura-2-parameter (K2P) evolutionary model (Kimura 1980) using MEGA11 (Tamura et al. 2021), with pairwise deletion of ambiguous sites. Node support was assessed by the bootstrap method with 1000 replicates. The tree topology was visualised using FigTree v.1.4.4 (Rambaut 2009). In order to assign our specimens to molecular operational taxonomic units (MOTU), a species delimitation analysis was performed using the Assemble Species by Automatic Partitioning (ASAP) method (Puillandre et al. 2021). ASAP was run under the K2P model and the first partition was chosen according to the best ASAP score. Based on species delimitation results, the sequences assigned to the MOTU including the Mediterranean Dendostrea sp. specimens constitute Dataset 2 (DS2). Six sequences with > 5% of ambiguous bases were filtered out from DS2 (Suppl. material 1), which reduced its final size to 84 sequences. Phylogenetic relationships amongst haplotypes of DS2 were inferred using the Median-Joining (MJ) network method (Bandelt et al. 1999), as implemented in PopART v.1.7 (Leigh and Bryant 2015), with the parameter ε set to zero. Main haplogroups (i.e. clusters of closely-related haplotypes) within the MJ network were mapped across sampling localities in QGIS v.3.30.2 to disclose potential phylogeographic patterns. Sampling sites closer than 8 km were shown as a single sample in the map, but a single site haplotype map is available in Suppl. material 2. For each region and for the whole Mediterranean Sea, the number of haplotype (N), Nei’s haplotype diversity (h) and nucleotide diversity (π) were assessed using the package pegas v.1.3 on R v.4.3.1. To account for uneven sampling size between regions, standardised estimates of h, π and haplotype richness (H) were obtained following the re-sampling procedure described in Salvi et al. (2013) on Python v.3.10.11 using 1000 iterations and a re-sampling size of eight for the comparison between Mediterranean regions and between 281 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera the Mediterranean Sea and Rodrigues. The 95% confidence intervals of diversity estimates were computed by bootstrap re-sampling using the Python module SciPy v.1.15.2 with 1,000 replicates under the ‘basic’ method. Haplotype accumulation curves of the entire Mediterranean Sea, Rhodes and Cyprus were calculated using the function haploAccum() from the R package ape v.5.7-1 with 1000 iterations under the “random” method. For the whole Mediterranean Sea, the Chao1 asymptotic estimator for haplotype richness (Chao 1984) was assessed using the R package iNEXT (Hsieh et al. 2016). Finally, to assess population genetic structure between regions within the Mediterranean Sea (Table 1), Rodrigues was filtered out of DS2 and the 82 sequences left were used for the analysis of molecular variance (AMOVA; Excoffier et al. (1992)) with the R package poppr v.2.9.6. The components of covariance were tested with the function randtest() from the package ade4 v.1.7-22 with 1,000 permutations (Excoffier et al. 1992). Table 1. Geographical information on the sequences of Dendostrea cf. crenulifera and D. sandvichensis. Details are given on the 96 sequences of Dendostrea cf. crenulifera assigned to the MOTU “Ost21” (dataset DS2) and the sequences of Dendostrea sandvichensis from Île d’Ambre. The locality codes refer to the sample codes used in Fig. 2B. Country Region Localities (code) n Greece Astypalaia Astypalaia (AST)† 2 Greece Rhodes West Ákra Mérmigka (RHO0)† 2 Türkiye Türkiye Olympos (TUR1)† 2 Türkiye Türkiye Kekova (TUR2)† 2 Greece Rhodes East Paralia Antoni Kouin (RHO12) 6 Greece Rhodes East West of Touristiko Limani (RHO12) 1 Greece Rhodes East Agia Marina Beach (RHO12) 3 Greece Rhodes East Jordan Beach (RHO12) 2 Greece Rhodes West Kοpria Beach (RHO5) 5 Greece Rhodes West Port of Fanes (RHO6) 4 Greece Rhodes West Kerameni Beach (RHO7) 2 Greece Rhodes East St Paul’s Bay (RHO8) 7 Greece Rhodes East West of Prasonisi Beach (RHO13) 2 Greece Rhodes East South of Mavros Kavos Beach (RHO13) 5 Cyprus Cyprus Zygi Marina (CYP17) 2 Cyprus Cyprus West of Cape Dolos (CYP17) 3 Cyprus Cyprus St Raphael Marina (CYP17) 4 Cyprus Cyprus Polis Municipal Beach (CYP14) 6 Cyprus Cyprus Akrotiri Peninsula (CYP15) 1 Cyprus Cyprus Turtle Beach (CYP16) 1 Israel Israel/Lebanon West of Rosh HaNikra Islands (ISR1) 14 Israel Israel/Lebanon Ashqelon (ISR2) 9 Lebanon Israel/Lebanon Tyre (LEB) 3 Mauritius Rodrigues Passage Grand Bassin (ROD1) 5 Mauritius Rodrigues Baie Nord (ROD2) 3 Mauritius Île d’Ambre Bassin Trou Polite 8 n: number of sequences; †: GenBank sequences (KJ946446–KJ946453). 282 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Morphological assessment Shell morphology of specimens from the Mediterranean, Île d’Ambre and Rodrigues was assessed under a microscope. Photos of selected specimens were recorded using a Nikon D750 camera either connected to a Leica Z6 stereomicroscope or mounting a 60 mm 1:1 macro lens. All photographs were stacked using Helicon Remote and rendered using Helicon Focus. Based on molecular results, the shells were compared with the type materials of D. crenulifera and D. sandvichensis, as well as with the specimens illustrated in Oliver et al. (2004). The main morphological characteristics considered in the assessment included the attachment area (hinge), the shape of upper and lower valves, the shape of margins, the types of chomata and the inner colouration of valves. Results Phylogenetic relationships The dataset DS1 included 518 sequences and 649 aligned positions (base pairs, bp), amongst which 306 were variable (47.1%) and 253 were phylogenetically informative (39%). Twenty-nine MOTUs were identified amongst the 518 sequences of Ostreinae analysed (Fig. 1A), based on the lowest-score ASAP partition (score = 7.0) chosen according to the procedure described in Puillandre et al. (2021). Nine MOTUs were represented by a single sequence. Amongst the remaining 20 MOTUs, 16 received very high bootstrap support on the Neighbour-Joining tree (bootstrap, BS ≥ 95), three received good support (95 > BS ≥ 80) and one received low support (BS = 66). All the 89 Mediterranean Dendostrea sp. specimens clustered in a single well-supported MOTU named “Ost21” (BS = 99; ASAP p-value = 8.76e-01; Fig. 1). This MOTU also includes eight sequences of Dendostrea sp. from Türkiye and Greece from Crocetta et al. (2015), as well as sequences from Rodrigues. The MOTU “Ost21” is highly divergent (K2P-distance > 8%) from its closest sister clade “Ost18-Ost19-Ost20” (Fig. 1). This sister clade “Ost18-Ost19-Ost20” refers to sequences of specimens from south China and Malaysia (i.e. southern Chinese Sea) with unresolved nomenclature (Suppl. material 3). Within MOTU “Ost21”, there is no geographic structure with sequences from different Mediterranean regions admixed in multiple sub-clades and with Rodrigues sequences nested within them (Fig. 1). Intra-clade genetic distances (K2P-distances) within the MOTU “Ost21” range from 0% to 3.35% (p-distance = 3.25%), with an average of 1.53% (p-distance = 1.50%). Mean genetic distance (K2P-distance) between Mediterranean and Rodrigues specimens is 1.74% (p-distance = 1.70%). The eight sequences of Dendostrea specimens from Île d’Ambre clustered in a distinct MOTU named “Ost14” together with topotypic specimens of Dendostrea sandvichensis from Hawaii plus other sequences from the Pacific Ocean and the Caribbean Sea (BS = 99; ASAP p-value = 4.00e-01; Fig. 1). Within this clade, genetic distances (K2P-distances) range from 0 to 4.68% (p-distance = 4.47%), with an average of 1.58% (p-distance = 1.55%). Mean genetic distance (K2P-distance) between Île d’Ambre and Rodrigues specimens is 19.4% (p-distance = 16.8%). 283 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Figure 1. Neighbour-Joining (NJ) phylogenetic tree, based on the Ostreinae dataset (DS1) under the K2P evolutionary model. A. Complete NJ tree; B. Uncollapsed clade of the MOTU “Ost21” with sequences coloured, based on their respective geographical origin as reported in the legend. Bootstrap support values (BS) based on 1000 replicates are reported on nodes as dark grey (BS ≥ 95), light grey (95 > BS ≥ 80) or white (BS < 80) circles. Putative species clusters were collapsed, based on the results of the species delimitation analysis (Assemble Species by Automatic Partitioning; ASAP). All specimen of Dendostrea cf. crenulifera from the Mediterranean Sea and Rodrigues belong to the cluster “Ost21”, that has been highlighted in red. 284 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Phylogeographic structure and genetic diversity The dataset DS2 included 96 sequences and 649 aligned positions (base pairs, bp). The phylogenetic network includes 51 haplotypes over 96 sequenced specimens, with 43 haplotypes found only in the Mediterranean Sea and eight haplotypes found exclusively in Rodrigues (Fig. 2 and Suppl. material 1). Mediterranean haplotypes cluster in three groups, Rodrigues haplotypes forming a single cluster (indicated with distinct colours in Fig. 2). These four haplogroups showed shallow phylogenetic divergence that does not reflect geographic distance: two Mediterranean clusters and the Rodrigues cluster are separated by seven mutational steps one from each other, whereas they are separated by 13 mutational steps from the third Mediterranean cluster (green in Fig. 2). The phylogeographic pattern within the Mediterranean has no geographic structure, with the three haplogroups co-occurring on all sampling regions (Türkiye, Rhodes, Cyprus, Israel) and being syntopic in most sampling sites (Fig. 2). This observation is supported by the AMOVA, which revealed no significant structure either at the regional or population levels (Table 2). Haplotype diversity was remarkably high both in Rodrigues (observed h = 1 and N = 8) and in the eastern Mediterranean Sea, both as a whole (observed: h = 0.968 and N = 43; estimated: h = 0.969 and H = 7.227) and for single Eastern sampling regions (observed: h ranged from 0.833 to 0.978 and N from 1 to 19; estimated: h ranged from 0.945 to 0.979 and H from 6.446 to 7.413), as reported in Table 3. Nucleotide diversity was relatively high in the eastern Mediterranean Sea as a whole (π = 1.463 × 10-2) as well as for single Mediterranean sampling regions (π ranged from 1.377 × 10-2 to 1.591 × 10-2) as compared to Rodrigues (π = 0.480 × 10-2; Table 3). The haplotype accumulation curves do not reach an asymptote for any region (Cyprus, Rhodes and Israel/Lebanon), nor for the eastern Mediterranean as a whole (Fig. 3). In the Mediterranean Sea, 28 out of the 43 haplotypes found are singletons (> 50%) and the Chao1 estimator revealed an asymptotic value of haplotype richness of 98.32 (Fig. 3), suggesting that the true haplotype diversity of Dendostrea sp. is likely higher than estimated. Morphological assessment Despite molecular data indicating that the assayed oysters from the Mediterranean, Rodrigues and Île d’Ambre belong to two distinct MOTUs (“Ost21” for Mediterranean and Rodrigues and MOTU “Ost14” for Île d’Ambre), morphological assessment indicates that oysters from these three regions are morphologically very similar, with only slight differences that have no diagnostic value (Fig. 4). Indeed, the majority of shells analysed here have a large attachment area, the margin is irregularly toothed and the ribs are low, rounded and often obscure. This is the form more typical of Dendostrea sandvichensis that currently includes D. crenulifera from the Red Sea. The Mediterranean and Rodrigues shells are very variable, but the expression of the external ribbing is very poor in both, whereas the Île d’Ambre shells are often distinctly ribbed. Some shells have a reddish-black margin in both populations, but not in Île d’Ambre. The crenulations are numerous and angulate in both, whereas the Île d’Ambre shells appear to have more rounded crenulations. 285 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera In Mediterranean and Rodrigues shells, simple chomata are sparse and prominent, respectively, on the posterior dorsal margin. On the contrary, in Île d’Ambre shells, simple chomata are prominent all along the shell. Pustular (lophine) chomata are present between crenulations and on posterior margin only in the Mediterranean shells, whereas are sparse or absent in Rodrigues and Île d’Ambre shells. Figure 2. Phylogeography of Dendostrea cf. crenulifera from the Mediterranean Sea and Rodrigues. A. Median-Joining (MJ) haplotype network, based on the MOTU “Ost21” dataset (DS2). The four closely-related haplogroups within the MJ network are coloured; B. Their geographical distribution is reported on the map. Sampling sites closer than 8 km were shown as a single sample in the map, but a single site haplotype map is available in Suppl. material 2. The colouring of haplogroups is independent from the colouring of the sequences’ origin used in Figs 1, 3. For each locality, the sampling size is reported (n). 292 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Conclusion This study highlights the effectiveness of molecular methods for reliable identification of non-indigenous species and for the reconstruction of the invasion process. Mitochondrial data are particularly well-suited as an initial approach for both applications. COI, in particular, is the most commonly used barcode in animal studies and offers the largest reference library in public repositories, which is crucial for reliable species identification. In terms of invasion history, mitochondrial markers have the advantage of being highly susceptible to genetic drift, such as bottlenecks, due to the fourfold difference in effective population sizes between the haploid, uniparentally inherited mitochondrial genome and the nuclear genome (Hudson and Turelli 2003). By including specimens from the western Indian Ocean, we resolved the long-standing taxonomic uncertainty surrounding the small non-indigenous oysters invading the Mediterranean Sea, assigning them to Dendostrea cf. crenulifera. By combining data on geographic patterns of genetic diversity in the non-native range with species traits and characteristics (both biotic and abiotic), we gathered crucial information on dispersal mode, propagule pressure and temporal and spatial patterns of establishment, allowing us to dissect the invasion stages from arrival to post-establishment spread in the Mediterranean Sea. The global trend of increasing sea surface temperature and salinity in the eastern Mediterranean Sea likely facilitated the spread of this non-indigenous oyster (Raitsos et al. 2010; Albano et al. 2021a). The invasibility of the eastern Mediterranean Sea is determined by dynamic processes that may vary over time (Daly et al. 2023). As global warming continues, an extended decline in native species is expected (Albano et al. 2021a), potentially creating suitable environmental conditions and empty niche opportunities for D. cf. crenulifera in the eastern Mediterranean Sea, as well as at the western invasion front towards the Aegean and central Mediterranean Seas. This could pose a threat to native Mediterranean species such as Ostrea stentina and Ostrea edulis (Daly et al. 2023). Temporal data and molecular evidence suggest that ongoing propagule pressure facilitated the establishment and spread of this non-indigenous species, revealing a possible time lag between these two stages of the invasion. Such a time lag is well documented in other Lesspesian species. For example, in the mussel Brachidontes pharaonis, there was an extensive time lag between introduction and the climate-warming-driven spread 120 years later (Rilov et al. 2004). Whether the time lag observed for D. cf. crenulifera can be attributed to the genetic admixture of different evolutionary lineages in the Mediterranean and the introduction of adaptative genetic variation for new areas and habitats deserves further research. Additionally, further research in the Red Sea is necessary to validate the assignment of Mediterranean Dendostrea to D. crenulifera using topotypes and to precisely identify the source area(s), helping to distinguish between single vs. multiple source hypotheses. A good understanding of the introduction process is important for evaluating the impact on Mediterranean ecosystems and developing mitigation strategies. Acknowledgements Delcour N. would like to acknowledge the University of L’Aquila. We thank Jonathan Belmaker and Shahar Malamud, Tel Aviv University and Carlos Jiménez, Enalia Physis Environmental Research Centre and Niki Chartosia, University of Cyprus, for facilitating field activities in Israel and Cyprus, respectively. 293 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding The collection of samples from Israel and Cyprus used in this work was supported by the projects “Historical ecology of Lessepsian migration” (Austrian Science Fund, P28983-B29, to PGA) and “Drivers of biodiversity loss in the Eastern Mediterranean” (Austrian Science Fund, P 34509-B, to PGA and M. Zuschin). Author contributions Daniele Salvi and Matteo Garzia contributed to the study conception and design. All authors performed sample collection and preparation. Genetic data collection and analysis were performed by Matteo Garzia, Nathan Delcour and Daniele Salvi. The first draft of the manuscript was written by Nathan Delcour, Matteo Garzia and Daniele Salvi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Author ORCIDs Nathan Delcour https://orcid.org/0000-0001-8203-9301 Matteo Garzia https://orcid.org/0000-0002-0918-9925 P. Graham Oliver https://orcid.org/0000-0002-9481-0711 Emanuele Berrilli https://orcid.org/0000-0001-8081-8600 Andrea Toso https://orcid.org/0000-0001-6252-924X Michel Bariche https://orcid.org/0000-0001-6831-4311 Paolo G. Albano https://orcid.org/0000-0001-9876-1024 Paolo Mariottini https://orcid.org/0000-0003-1044-7108 Daniele Salvi https://orcid.org/0000-0002-3804-2690 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Al-Kandari M, Oliver PG, Salvi D (2021) Molecular and morphological systematics of a new, reef forming, cupped oyster from the northern Arabian Gulf: Talonostrea salpinx new species. ZooKeys 1043: 1–20. https://doi.org/10.3897/zookeys.1043.66992 Albano PG, Gallmetzer I, Haselmair A, Tomašových A, Stachowitsch M, Zuschin M (2018) Historical ecology of a biological invasion: The interplay of eutrophication and pollution determines time lags in establishment and detection. 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Diversity & Distributions 18: 1042–1055. https://doi.org/10.1111/j.14724642.2012.00894.x 301 NeoBiota 101: 277–302 (2025), DOI: 10.3897/neobiota.101.154917 Nathan Delcour et al.: High diversity in the Eastern Mediterranean oyster Dendostrea aff. crenulifera Supplementary material 1 Detailed information on the 96 sequences of Dendostrea cf. crenulifera and D. sandvichensis Authors: Nathan Delcour, Matteo Garzia, Daniele Salvi Data type: ods Explanation note: Information concerns the sequences of Dendostrea cf. crenulifera assigned to the MOTU “Ost21” (dataset DS2), and the sequences of Dendostrea sandvichensis from Île d’Ambre. Specimens and GenBank codes and coordinates of each sampling locality are reported. Inventory numbers (Inv. numb.) are provided for the dry shells deposited in the following collections: Roma Tre University (referent: Prof. Paolo Mariottini, “BAU”), the Malacological Collection of the University of L’Aquila (“OS”), the Stazione Zoologica Anton Dohrn (“SZN”), the Natural History Museum in Vienna (“112930‐LM”) and the National Museum of Wales (“NMW”). Haplotype codes are reported only for the sequences used in the Median-Joining (MJ) network (see Fig. 2B of the main article). The locality codes refer to the site codes used in Suppl. material 2B. †: sequences retrieved from GenBank; ‡: sequences filtered out from the MJ analysis because they contain > 5% of ambiguous sites; §: sequences of specimens identified as Dendostrea sandvichensis. Copyright notice: 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. Link: https://doi.org/10.3897/neobiota.101.154917.suppl1 Supplementary material 2 Phylogeography of Dendostrea cf. crenulifera from the Mediterranean Sea and Rodrigues Authors: Nathan Delcour, Matteo Garzia, Daniele Salvi Data type: png Explanation note: A Median-Joining (MJ) haplotype network, based on the MOTU “Ost21” dataset (DS2). The four closely-related haplogroups within the MJ network are coloured and B their detailed geographical distribution is reported on the map, based on each sampling site. The colouring of haplogroups is independent from the colouring of the sequences’ origin used in Figs 1, 3. For each locality, the sampling size is reported (n). Copyright notice: 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. Link: https://doi.org/10.3897/neobiota.101.154917.suppl2