INTRODUCTION
⌅In the last few decades, the number of alien species in the Mediterranean Sea has been constantly increasing (Zenetos and Galanidi 2020ZenetosA., GalanidiM.2020. Mediterranean non indigenous species at the start of the 2020s: recent changes. Mar. Biodivers. Rec.13: 10. 10.1186/s41200-020-00191-4). However, the estimates rely heavily on correct identifications of alien species, a task that is often challenging, especially for taxa that show a high level of phenotypic plasticity and for cryptic species complexes (Faulwetter et al. 2017FaulwetterS., SimbouraN., KatsiarasN., ChatzigeorgiouG., ArvanitidisC.2017. Polychaetes of Greece: an updated and annotated checklist. Biodivers. Data J.e20997. 10.3897/BDJ.5.e20997, Galià-Camps et al. 2020Galià-CampsC., CarmonaL., CabritoA., et al. 2020. Double trouble. A cryptic first record of Berghia arinae Carmona, Pola, Gosliner, and Cervera 2014 in the Mediterranean Sea. Mediterr. Mar. Sci.21: 191-200. 10.12681/mms.20026, Garzia et al. 2022GarziaM., FurfaroG., RendaW., et al. 2022. Mediterranean spreading of the bicolor purse oyster, Isognomon bicolor, and the chicken trigger, Malleus sp., vs. the Lessepsian prejudice. Mediterr. Mar. Sci.23: 777-788. 10.12681/mms.29218). Lack of morphological diagnostic characters is frequent in marine bivalves, which account for more than 50 alien species established in the Mediterranean Sea (Zenetos et al. 2022ZenetosA., AlbanoP.G., GarciaE.L., et al. , 2022. Established non-indigenous species increased by 40% in 11 years in the Mediterranean Sea. Mediterr. Mar. Sci.23. 10.12681/mms.29106). Incorrect identifications of alien species could misestimate the number of these taxa and provide a biased picture of their non-native distribution (Zenetos et al. 2017ZenetosA., ÇinarM.E., CrocettaF., et al. 2017. Uncertainties and validation of alien species catalogues: The Mediterranean as an example. Estuar. Coast. Shelf Sci.191: 171-187. 10.1016/j.ecss.2017.03.031).
The species Pinctada fucata (A. Gould, 1850) and Pinctada radiata (Leach, 1814) are considered alien species in the Mediterranean Sea (Scuderi et al. 2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426, Zenetos and Galanidi 2020ZenetosA., GalanidiM.2020. Mediterranean non indigenous species at the start of the 2020s: recent changes. Mar. Biodivers. Rec.13: 10. 10.1186/s41200-020-00191-4) and belong to the species complex P. fucata/imbricata/radiata (Margaritidae Blainville, 1824), whose members share similar morphological characters that make their identification challenging. According to the phylogenetic analyses by Tëmkin (2010TëmkinI.2010. Molecular phylogeny of pearl oysters and their relatives (Mollusca, Bivalvia, Pterioidea). BMC Evol. Biol.10: 342. 10.1186/1471-2148-10-342) based on three nuclear markers (28S, ITS2, and H3) and one mitochondrial (16S) marker, P. fucata/imbricata/radiata are distinct clades but show low phylogenetic divergence. These clades are interpreted as three valid species with quite distinct geographical distributions by Cunha et al. (2011CunhaR.L., BlancF., BonhommeF., Arnaud-HaondS.2011. Evolutionary Patterns in Pearl Oysters of the Genus Pinctada (Bivalvia: Pteriidae). Mar. Biotechnol.13: 181-192. 10.1007/s10126-010-9278-y) based on the mitochondrial marker cox1 and the nuclear marker 18S: P. imbricata Röding, 1798 from the western Atlantic Ocean; P. fucata from the Indo-Pacific; and P. radiata from the eastern Indian Ocean and the Red Sea. This classification into three species is currently accepted by MolluscaBase (2023MolluscaBase eds. 2023. MolluscaBase. Pinctada Röding, 1798. Accessed through: World Register of Marine Species at: https://www.marinespecies.org/aphia.php?p=taxdetails&id=138396 on 2024-04-23).
However, while molecular data have resolved the systematics of this species complex, the morphological identification of pearl oysters recorded in the Mediterranean Sea is still debated. First records of pearl oysters in the Mediterranean Sea were morphologically assigned to Pinctada imbricata radiata (Leach, 1814) (Doğan and Nerlović 2008DoğanA., NerlovićV.2008. On the occurrence of Pinctada radiata (Mollusca: Bivalvia: Pteriidae), an alien species in Croatian waters. Acta Adriat. 49: 155-158., Mienis 2004MienisH. K.2004. New data concerning the presence of Lessepsian and other Indo-Pacific migrants among the molluscs in the Mediterranean Sea with emphasis on the situation in Israel. Turk. J. Aquat. Life2: 117-131., Zenetos et al. 2004ZenetosA., GofasS., RussoG., TempladoJ.2004. CIESM Atlas of Exotic Species in the Mediterranean, Vol 3 – Molluscs.) and later to P. radiata (e.g. Bellaaj-Zouari et al. 2012Bellaaj-ZouariA., DkhiliS., GharsalliR., et al. 2012. Shell morphology and relative growth variability of the invasive pearl oyster Pinctada radiata in coastal Tunisia. J. Mar. Biol. Assoc. U.K.92: 553-563. 10.1017/S0025315411001925, Petović and Mačić 2018Petović S., Mačić V. 2018. New data on Pinctada radiata (Leach, 1814) (Bivalvia: Pteriidae) in the Adriatic Sea. Acta Adriat. 58: 357-361. 10.32582/aa.58.2.14) following the taxonomic revision of the complex (Cunha et al. 2011CunhaR.L., BlancF., BonhommeF., Arnaud-HaondS.2011. Evolutionary Patterns in Pearl Oysters of the Genus Pinctada (Bivalvia: Pteriidae). Mar. Biotechnol.13: 181-192. 10.1007/s10126-010-9278-y). Barbieri et al. (2016BarbieriM., DeidunA., MaltagliatiF., CastelliA.2016. A contribution to the phylogeography of Pinctada imbricata radiata (Leach, 1814) (Bivalvia: Pteriidae) from the Eastern Mediterranean Sea by means of the mitochondrial COI marker. Ital. J. Zool.83: 113-120.10.1080/11250003.2015.1106012) confirmed that all the sequenced specimens collected in the Mediterranean Sea belong to P. radiata. After this work, recent reports indicated the rapid spread of the alien P. radiata from the southern-central and eastern Mediterranean, where this species is considered well-established, towards the western Mediterranean (Ballesteros et al. 2020BallesterosE., MarsinyachE., BagurM., et al. , 2020. The pearl oyster Pinctada imbricata radiata (Leach, 1814) (Bivalvia: Pteriidae) reaches Minorca, Balearic Islands. Boll. Soc. Hist. Nat. Balears63., Derbali et al. 2019DerbaliA., KandeelK.E.JarbouiO.2019. Comparison of the Dynamics between Coastal and Midshore Populations of Pinctada radiata (Leach, 1814) (Mollusca: Bivalvia) in the Gulf of Gabes, Tunisia. Turk. J. Fish. Aquat. Sci.20: 301-310. 10.4194/1303-2712-v20_4_06, Gavrilović et al. 2017GavrilovićA., PiriaM., GuoX.-Z., et al. 2017. First evidence of establishment of the rayed pearl oyster, Pinctada imbricata radiata (Leach, 1814), in the eastern Adriatic Sea. Mar. Pollut. Bull.125: 556-560. 10.1016/j.marpolbul.2017.10.045, Petović and Mačić 2018Petović S., Mačić V. 2018. New data on Pinctada radiata (Leach, 1814) (Bivalvia: Pteriidae) in the Adriatic Sea. Acta Adriat. 58: 357-361. 10.32582/aa.58.2.14, Png-Gonzalez et al. 2021Png-GonzalezL., Aguilo-ArceJ., Vázquez-LuisM., CarbonellA.2021. New occurrence of Pinctada imbricata radiata (Leach, 1814) in the Balearic Archipelago (NW Mediterranean Sea). BioInvasions Rec. 10: 853-858. 10.3391/bir.2021.10.4.09). Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) and Cunningham Aparicio and Méndez (2021Cunningham AparicioB., MéndezI.2021. First record of the potentially invasive Pinctada imbricata fucata (Gould, 1850) (Bivalvia Pteriidae) along the Mediterranean coasts of Spain. Biodivers. J.12: 585-588. 10.31396/Biodiv.Jour.2021.12.3.585.588) claimed that another Lessepsian species, P. fucata, occurs throughout the Mediterranean Sea, in many cases in nearby localities with P. radiata. However, these studies are based on only morphological characters. According to Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426), P. fucata and P. radiata can be adequately discriminated by morphological characters. However, their diagnostic value has not been verified with molecular data. Despite the lack of molecular evidence, the most recent checklists have included both P. fucata and P. radiata as present in Italian waters (Renda et al. 2022RendaW., AmatiB., BogiC., et al. 2022. The new Checklist of the Italian Fauna: marine Mollusca. Biogeogr. J. Integr. Biogeogr.37: ucl005. 10.21426/B637156028) and in the Mediterranean Sea (Zenetos et al. 2022ZenetosA., AlbanoP.G., GarciaE.L., et al. , 2022. Established non-indigenous species increased by 40% in 11 years in the Mediterranean Sea. Mediterr. Mar. Sci.23. 10.12681/mms.29106).
In this study, we performed an integrative assessment based on morphological and genetic data on pearl oysters collected across the central, southern and eastern Mediterranean Sea. Molecular species delimitation analyses based on the barcode marker cox1 allowed a straightforward taxonomic assignment of collected specimens. Based on these results, we tested the diagnostic value of the morphological characters proposed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426). Finally, we provided an updated taxonomic classification and distribution of pearl oysters invading the Mediterranean Sea.
MATERIAL AND METHODS
⌅Sampling areas
⌅Pearl oysters were sampled between 2011-2021 in nine localities from the central, southern and eastern Mediterranean Sea. These also include localities where both P. fucata and P. radiata were previously recorded (Scuderi et al. 2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426). Details of all the sampling localities are listed in Table 1. All tissues of Pinctada specimens are stored in ethanol 95%. All dry shells are in the molluscan collection of Paolo Mariottini at the Roma Tre University, Rome, Italy.
Molecular analyses
⌅A piece of adductor muscle was clipped and total DNA extraction was carried out using the high-salt protocol (Evans 1990EvansG.A.1990. Molecular Cloning: A Laboratory Manual. Second edition. Volumes 1, 2, and 3. Current Protocols in Molecular Biology. Volume 1 and 2. Cells61: 17-18. 10.1016/0092-8674(90)90210-6). The standard barcoding fragment cox1 was amplified using universal primers LCO1490 and HCO2198 (Folmer et al. 1994FolmerO., BlackM., HoehW., et al. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol.3: 294-299.). The PCR conditions used were a 3 min denaturation step at 94°C; 35 cycles of 94°C/60 s, 48°C/60 s, 72°C/60 s; and a 10 min final extension at 72°C. The amplified products were sequenced by Macrogen Inc., Amsterdam, the Netherlands. The sequences are deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/GenBank/) with accession numbers OR676328- OR676344.
We downloaded 263 cox1 sequences of Pinctada spp. (Margaritidae) from GenBank (the GB dataset, updated to 31/12/2022) to build a dataset of 281 cox1 sequences including the 17 newly generated sequences of Pinctada sp. and the outgroup Pteria hirundo (Linnaeus, 1758) (GB Accession Number AF120647). This dataset was aligned using the G-INS-i algorithm in the MAFFT v.7 server (Katoh et al. 2019KatohK., RozewickiJ., YamadaK.D.2019. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 20: 1160-1166. 10.1093/bib/bbx108) and trimmed to the standard cox1 barcoding fragment (the 5′ portion of the gene) using Folmer’s primers as a reference.
Molecular species identification was based on distance-based and phylogenetic tree-based approaches. A preliminary phylogenetic assessment was performed with a neighbour-joining (NJ) tree based on uncorrected p-distance calculated in MEGA X (Kumar et al. 2018KumarS., StecherG., LiM., KnyazC., TamuraK.2018. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol.35: 1547-1549. 10.1093/molbev/msy096) and the pairwise deletion option. The NJ tree allowed us to detect 41 misidentified sequences named as P. margaritifera but belonging to the freshwater Margaritifera margaritifera (Linnaeus, 1758) [Margaritiferidae J. Henderson, 1929 (1910)]. These misidentified sequences were excluded and a final dataset of 222 sequences was used for distance-based and tree-based species delimitation analyses.
Assemble species by automatic partitioning (ASAP) analysis were run using the ASAP web interface [https://bioinfo.mnhn.fr/abi/public/asap; (Puillandre et al. 2021PuillandreN., BrouilletS., AchazG.2021. ASAP: assemble species by automatic partitioning. Mol. Ecol. Resour.21: 609-620. 10.1111/1755-0998.13281)] with the Kimura-two parameter [K2P; (Kimura 1980KimuraM.1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol.16: 111-120. 10.1007/BF01731581)] substitution model and default parameters. ASAP delimitation was defined by evaluating both the partitions with the first and the second-best ASAP scores according to Puillandre et al. (2021PuillandreN., BrouilletS., AchazG.2021. ASAP: assemble species by automatic partitioning. Mol. Ecol. Resour.21: 609-620. 10.1111/1755-0998.13281).
As a tree-based method, we used the multirate Poisson tree processes (mPTP) model (Kapli et al. 2017KapliP., LutteroppS., ZhangJ., et al. 2017. Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo. Bioinformatics33: 1630-1638. 10.1093/bioinformatics/btx025) based on a maximum likelihood (ML) tree. ML analyses were performed in the W-IQ-TREE web server v.1.6.12 [http://iqtree.cibiv.univie.ac.at/; (Trifinopoulos et al. 2016TrifinopoulosJ., NguyenL.T., von HaeselerA., MinhB.Q.2016. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 44: W232-235. 10.1093/nar/gkw256)]. Node support was assessed with 1000 pseudoreplicates of ultrafast bootstrapping [uBS; (Minh et al. 2013MinhB.Q., NguyenM.A.T., von HaeselerA.2013. Ultrafast Approximation for Phylogenetic Bootstrap. Mol. Biol. Evol.30: 1188-1195. 10.1093/molbev/mst024)]. FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the tree. The previous ML tree was used as an input file (excluding the outgroup) for mPTP analysis using the web platform (https://mptp.h-its.org/#/tree).
Furthermore, we performed a phylogeographic analysis of pearl oysters’ haplotype across the native and non-native (Mediterranean) distribution ranges, using phylogenetic network methods based on 57 cox1 sequences assigned to the Pinctada fucata/imbricata/radiata species complex. We used POPART v1.7 (Leigh and Bryant 2015LeighJ.W., BryantD.2015. popart: full-feature software for haplotype network construction. Methods Ecol. Evol.6: 1110-1116. 10.1111/2041-210X.12410) to build phylogenetic networks based on the median-joining method (Bandelt et al. 1999BandeltH.J., ForsterP., RöhlA.1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol.16: 37-48. 10.1093/oxfordjournals.molbev.a026036) and the statistical parsimony method TCS (Clement et al. 2000ClementM., PosadaD., CrandallK.A.2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol.9: 1657-1659. 10.1046/j.1365-294x.2000.01020.x). We visualized the geographic distribution of haplotypes by assigning them different colours according to the region of origin.
Finally, we mapped all the occurrences of specimens molecularly identified as P. radiata (Barbieri et al. 2016BarbieriM., DeidunA., MaltagliatiF., CastelliA.2016. A contribution to the phylogeography of Pinctada imbricata radiata (Leach, 1814) (Bivalvia: Pteriidae) from the Eastern Mediterranean Sea by means of the mitochondrial COI marker. Ital. J. Zool.83: 113-120.10.1080/11250003.2015.1106012, Gavrilović et al. 2017GavrilovićA., PiriaM., GuoX.-Z., et al. 2017. First evidence of establishment of the rayed pearl oyster, Pinctada imbricata radiata (Leach, 1814), in the eastern Adriatic Sea. Mar. Pollut. Bull.125: 556-560. 10.1016/j.marpolbul.2017.10.045, this study) along with Sardinian specimens morphologically identified as P. radiata [Stasolla et al. 2014StasollaG., RioloF., MacaliA., et al. 2014. Further spreading in the Italian seas of already established non-indigenous mollusc species. Mar. Biodivers. Rec.7: e120. 10.1017/S1755267214001079, Grech and Caracciolo (2023) in Fortic et al. (2023ForticA., Al-Sheikh RasheedR., AlmajidZ., et al. 2023. New records of introduced species in the Mediterranean Sea (April 2023). Mediterr. Mar. Sci.24: 182-202. 10.12681/mms.34016)] and specimens identified either as P. fucata or P. radiata by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) and Cunningham Aparicio and Méndez (2021Cunningham AparicioB., MéndezI.2021. First record of the potentially invasive Pinctada imbricata fucata (Gould, 1850) (Bivalvia Pteriidae) along the Mediterranean coasts of Spain. Biodivers. J.12: 585-588. 10.31396/Biodiv.Jour.2021.12.3.585.588) based on the characters proposed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426). Occurrences of specimens analysed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) are georeferenced according to the information on localities provided in Table 2 of their study.
| Species/specimens | S_OUTa | A_TOOb | H_LINc | L_AREd | Shell sculpture | Shell colour | Height of the shell | Environment |
|---|---|---|---|---|---|---|---|---|
| Pinctada radiata [Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426)] | 1 | 1 | 1 | 1 | Numerous rows of dense and pointed processes | Red-browndish with darker vertical strips | High up 75 mm | Hard substrate |
| Pinctada fucata [Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426)] | 2 | 2 | 2 | 2 | Low number of rows and sparse, blunt and wider processes | Greenish with paler vertical strips | high up to 45 mm | Sandy bottom near estuarine areas |
| RM3-PM-142a | 1 | 2 | 1 | 2 | Numerous rows of dense and pointed processes | Reddish with darker radial rows | 41 mm | On a buoy rope |
| RM3-PM-142b | 1 | 2 | 1 | 1 | Numerous rows of dense and pointed processes | Reddish with darker radial rows | 45 mm | On a buoy rope |
| RM3-PM-149a | 2 | 1 | 1 | 2 | Almost smooth; low number of rows of sparse processes | Light brownish with dark radial strips | 46 mm | On rocky substrate |
| RM3-PM-149b | 1 | 1 | 1 | 1 | Numerous rows of dense and pointed processes | Reddish with white radial strips | 45 mm | On rocky substrate |
| RM3-PM-153a | 1 | * | 1 | 2 | Numerous rows of dense and pointed processes | Yellowish with brownish radial strips | 47 mm | On rocky substrate |
| RM3-PM-153b | 1 | * | 1 | 2 | Numerous rows of dense and pointed processes | Reddish with white radial strips | 49 mm | On rocky substrate |
| RM3-PM-154a | 1 | 1 | 1 | 2 | Partially encrusted; dense and pointed processes visible only on the edge | Uniformly reddish | 42 mm | On rocky substrate |
| RM3-PM-154b | 1 | * | 1 | 2 | Almost smooth; low number of rows of sparse processes | Uniformly reddish | 44 mm | On rocky substrate |
| RM3-PM-171a | 1 | 2 | * | 1 | Uniformly smooth | Uniformly black | 41 mm | On rocky substrate |
| RM3-PM-171b | 1 | * | 1 | 1 | Uniformly smooth | Uniformly black | 43 mm | On rocky substrate |
| RM3-PM-173 | 1 | 1 | 2 | 1 | Numerous rows of dense and pointed processes | Light green with dark green radial strips | 58 mm | On rocky substrate |
| RM3-PM-174a | 1 | 1 | 2 | 1 | Numerous rows of dense and pointed processes | Green without radial strips | 54 mm | Beached in front of a sandy coast |
| RM3-PM-174b | 1 | 1 | * | 1 | Low number of rows of sparse and wider processes | Light green with dark green strips | 55 mm | Beached in front of a sandy coast |
| RM3-PM-175a | 2 | 1 | * | 2 | Numerous rows of dense and pointed processes | Dark brown reddish with white radial strips | 43 mm | On rocky substrate |
| RM3-PM-175b | 2 | 2 | * | 2 | Low number of rows of sparse processes | Reddish with white radial strips | 41 mm | On rocky substrate |
| RM3-PM-176a | 1 | 2 | 1 | 2 | Uniformly smooth | Encrusted | 75 mm | On rocky substrate |
| RM3-PM-176b | 1 | * | 1 | 2 | Uniformly smooth | Encrusted | 80 mm | On rocky substrate |
Morphological analysis
⌅To assess the diagnostic value of morphological characters proposed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426), we carried out a morphological assessment of the molecularly identified pearl oysters collected in the Mediterranean Sea. We analysed the following six morphological characters that are considered discriminant between P. fucata and P. radiata by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) (Table 2): (i) the general outline of the shell, (ii) the hinge structure (anterior tooth of the left valve), (iii) the shape of the ligament (hinge line), (iv) the extent of the ligament area, (v) the sculpture of the shell, and (vi) the prevalent colour of the valves. Characters’ states were assigned based on Scuderi et al (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426: Figures 6-7, 10-11, 17-18, 20-21, 27-30 and 32-33 and Table 1). To better visualize character state distribution across the analysed specimens, we coded states into numbers, except for the “shell sculpture” and the “shell colour” because of the wide variability of these two characters, for which a short description of the state is provided. In “shell sculpture”, the term “processes” used in Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) refers to growths of the outer shell similar to imbricated lamellar structures. State “1” and “2” in Table 2 refer respectively to the diagnostic characters of P. radiata and P. fucata sensu Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426). We also added information relative to the specimen size and environmental characteristics of collection sites that are considered by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) (Table 2). According to Table 2, we selected two couples of voucher specimens to illustrate the morphological characteristics deemed as diagnostic of P. radiata (RM3-PM-149b and RM3-PM-174b) and P. fucata (RM3-PM-175a and RM3-PM-175b) sensu Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426).
RESULTS
⌅Molecular analyses
⌅All species delimitation analyses, whether distance-based or tree-based, support the three clades P. fucata/imbricata/radiata as distinct species (Fig. 1). In the ML tree, all 17 specimens collected in the Mediterranean clustered within the clade of Pinctada radiata (Fig. 2), which received high bootstrap support (uBS=98). The P.radiata clade is sister to the clades of P. imbricata and P. fucata, which also received high support (uBS=99-100).
The median-joining network showed three well distinct haplotype groups corresponding to sequences of (i) P. imbricata from the Atlantic Ocean, (ii) P. fucata from the Indo-Pacific Ocean, and (iii) P. radiata from the Mediterranean Sea and Persian Gulf (Fig. 2). These three haplogroups were separated by a high number (21-30) of mutational steps (nucleotide substitutions) and were recovered as separated sub-networks in the statistical parsimony analysis (result not shown).
Morphological analysis
⌅Results of the screening of the six morphological characters proposed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) in the 17 specimens analysed in this study are summarized in Table 2. The 17 specimens, molecularly identified as P. radiata (Fig. 1), show a mixture of character states that were considered as diagnostic of either P. fucata or P. radiata by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426). Indeed, specimens of P. radiata show either an “oval” (Fig. 3G-H, J-K) or “rounded” (Fig. 3A-B, D-E) general outline, a “not duplicated” (Fig. 3L) or “duplicated” (Fig. 3C, F, I) hinge structure, a “straight” (Fig. S2c, S3c) or “curved” (Fig. 3C) ligament shape, and a “wide” (Fig. 3C, F) or “narrow” (Fig. 3I, L) ligament area. Furthermore, we observed intermediate states in the characters “hinge structure (A_TOO)” (Fig. S6, S13c, S14c) and “shape of the ligament (H_LIN)” (Fig. 3F, I, L). The variation of the sculpture and colours of the shells showed more than two alternative states described by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426; Table 1) for P. fucata and P. >radiata and included at least 7 and 12 states, respectively.
DISCUSSION
⌅The low resolution of systematic relationships and taxonomic status of the P. fucata/imbricata/radiata species complex was previously due to extensive variation in shell characters among and within populations and wide geographical distribution. Phylogenetic studies based on molecular data have accurately identified three evolutionary and taxonomic units within this complex, recovered as reciprocally monophyletic clades (Cunha et al. 2011CunhaR.L., BlancF., BonhommeF., Arnaud-HaondS.2011. Evolutionary Patterns in Pearl Oysters of the Genus Pinctada (Bivalvia: Pteriidae). Mar. Biotechnol.13: 181-192. 10.1007/s10126-010-9278-y, Somrup et al. 2022SomrupS., SangsawangA., McMillanN., et al. 2022. Pinctada phuketensis sp. nov. (Bivalvia, Ostreida, Margaritidae), a new pearl oyster species from Phuket, western coast of Thailand. ZooKeys1119: 181-195. 10.3897/zookeys.1119.87724, Tëmkin 2010TëmkinI.2010. Molecular phylogeny of pearl oysters and their relatives (Mollusca, Bivalvia, Pterioidea). BMC Evol. Biol.10: 342. 10.1186/1471-2148-10-342) that are currently reported as distinct species in MolluscaBase (2023MolluscaBase eds. 2023. MolluscaBase. Pinctada Röding, 1798. Accessed through: World Register of Marine Species at: https://www.marinespecies.org/aphia.php?p=taxdetails&id=138396 on 2024-04-23). Results from the present study are in close agreement with these findings. Molecular species delimitation and phylogenetic network analyses corroborate the taxonomic status of the species P. fucata, P. imbricata and P. radiata (Fig. 1 and Fig. 2) and provide further evidence of the utility of the cox1 barcode marker in identifying specimens to these taxa.
All sequenced specimens collected in the Mediterranean Sea from 16 different localities, either in this study or in previous studies (Barbieri et al. 2016BarbieriM., DeidunA., MaltagliatiF., CastelliA.2016. A contribution to the phylogeography of Pinctada imbricata radiata (Leach, 1814) (Bivalvia: Pteriidae) from the Eastern Mediterranean Sea by means of the mitochondrial COI marker. Ital. J. Zool.83: 113-120.10.1080/11250003.2015.1106012, Gavrilović et al. 2017GavrilovićA., PiriaM., GuoX.-Z., et al. 2017. First evidence of establishment of the rayed pearl oyster, Pinctada imbricata radiata (Leach, 1814), in the eastern Adriatic Sea. Mar. Pollut. Bull.125: 556-560. 10.1016/j.marpolbul.2017.10.045), are unambiguously assigned to P. radiata (Figs 1 and 2). Compared with previously available data, this study contributes nine additional records of P. radiata from the southeastern Mediterranean Basin as well as northern regions and provides the first molecular validation of the presence of this species in Sardinia (Stasolla et al. 2014StasollaG., RioloF., MacaliA., et al. 2014. Further spreading in the Italian seas of already established non-indigenous mollusc species. Mar. Biodivers. Rec.7: e120. 10.1017/S1755267214001079, Grech and Caracciolo 2023 in Fortic et al. 2023ForticA., Al-Sheikh RasheedR., AlmajidZ., et al. 2023. New records of introduced species in the Mediterranean Sea (April 2023). Mediterr. Mar. Sci.24: 182-202. 10.12681/mms.34016). Sardinian records indicate that this Lessepsian species is spreading further towards the northern and western sectors of the Mediterranean Sea. This concern is confirmed by recent molecular data, made available in GenBank at the time of preparation of our study, that indicate the presence of P. radiata also in the Adriatic Sea (Gavrilović et al. 2017GavrilovićA., PiriaM., GuoX.-Z., et al. 2017. First evidence of establishment of the rayed pearl oyster, Pinctada imbricata radiata (Leach, 1814), in the eastern Adriatic Sea. Mar. Pollut. Bull.125: 556-560. 10.1016/j.marpolbul.2017.10.045). All these records verified by molecular data, including the recent data from the Adriatic Sea, are mapped in Figure 4, which represents the latest update on the distribution and spreading of P. radiata in the Mediterranean Sea.
Distinguishing specimens of these three closely related species based on morphological characters remains challenging. Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) were the first to propose a set of six diagnostic morphological characters suitable to distinguish specimens of P. fucata and P. radiata collected from the Mediterranean Sea. However, our morphological assessment showed that molecularly identified specimens of P. radiata collected from several localities of the Mediterranean Sea encompass the entire suite of character states that Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) considered diagnostic of either P. fucata or P. radiata (Table 2). This finding is quite surprising considering the much lower number of specimens assessed morphologically in our study (N=17) compared with the study by Scuderi and colleagues (N=1284) and clearly suggests that the variation in shell colour and sculpture of P. radiata is vastly larger than that observed by this study. Another character that proved challenging in the morphological assessment was the shape of the hinge line. Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) described the hinge line either as straight or curved. We observed that in small specimens with an underdeveloped hinge region the hinge line seems straight or barely curved (see Supplementary Figs S2cd and S3cd), whereas in specimens with a more developed hinge area a well-defined curved hinge line occurs more frequently (see Supplementary Figs S5cd and S6cd). Therefore, while this character certainly has no taxonomic value, it seems that it may be related to the size variation of the hinge region.
The results of our combined and morphological assessments clearly demonstrated that the characters proposed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) have no taxonomic value. The morphological variability observed in the pearl oyster specimens from the Mediterranean Sea clearly represents the intraspecific variability of P. radiata (Bellaaj-Zouari et al. 2012Bellaaj-ZouariA., DkhiliS., GharsalliR., et al. 2012. Shell morphology and relative growth variability of the invasive pearl oyster Pinctada radiata in coastal Tunisia. J. Mar. Biol. Assoc. U.K.92: 553-563. 10.1017/S0025315411001925, Hmida et al. 2021HmidaL., FassatouiC., Missaoui S., et al. 2021. Morphological and genetic characterization of the invasive rayed pearl oyster Pinctada imbricata radiata (Mollusca: Bivalvia: Pteriidae) populations from contrasting environments along the Tunisian coast. Mar. Biol. Res.17: 200-214. 10.1080/17451000.2021.1919713). These findings are in agreement with previous morphometric investigations that documented an extensive variability of the shell morphology of P. radiata within populations in the native distribution area (Rajaei et al. 2014RajaeiM., PoorbagherH., FarahmandH., et al. 2014. Interpopulation differences in shell forms of the pearl oyster, Pinctada imbricata radiata (Bivalvia: Pterioida), in the northern Persian Gulf inferred from principal component analysis and elliptic Fourier analysis. Turk. J. Zool.38: 42-48. 10.3906/zoo-1207-9, 2015RajaeiM., FarahmandH., PoorbagherH., et al. 2015. Sympatric morphological and genetic differentiation of the pearl oyster Pinctada radiata (Bivalvia: Pterioida) in the northern Persian Gulf. J. Mar. Biol. Assoc. U. K.95: 537-543. 10.1017/S0025315414001611), as well as within non-native populations in the Mediterranean Sea (Bellaaj-Zouari et al. 2012Bellaaj-ZouariA., DkhiliS., GharsalliR., et al. 2012. Shell morphology and relative growth variability of the invasive pearl oyster Pinctada radiata in coastal Tunisia. J. Mar. Biol. Assoc. U.K.92: 553-563. 10.1017/S0025315411001925, Hmida et al. 2021HmidaL., FassatouiC., Missaoui S., et al. 2021. Morphological and genetic characterization of the invasive rayed pearl oyster Pinctada imbricata radiata (Mollusca: Bivalvia: Pteriidae) populations from contrasting environments along the Tunisian coast. Mar. Biol. Res.17: 200-214. 10.1080/17451000.2021.1919713). To date no diagnostic discrete morphological characters have been identified for populations of the closely related pearl oyster species P. imbricata, P. fucata and P. radiata (Huber 2010HuberM.2010. Compendium of bivalves. Vol. 1. ConchBooks., Ranson 1961RansonG.1961. Les especes d’huitres perlieres du genre Pinctada (Biologie de quelqes unes d'etre elles). Inst Roy Sci Nat Belg. Ser.67: 1-95.). In line with results from the present study, molecular data by Cunha et al. (2011CunhaR.L., BlancF., BonhommeF., Arnaud-HaondS.2011. Evolutionary Patterns in Pearl Oysters of the Genus Pinctada (Bivalvia: Pteriidae). Mar. Biotechnol.13: 181-192. 10.1007/s10126-010-9278-y) showed that morphological characters traditionally used to discriminate between P. fucata and P. martensii have no taxonomic value and represent the intraspecific variability of the P. fucata. Therefore, molecular validation remains essential for the identification of pearl oysters. In this respect, we have no evidence that P. fucata occurs in the Mediterranean Sea. Indeed, the Mediterranean records of P. fucata (Cunningham Aparicio and Méndez 2021Cunningham AparicioB., MéndezI.2021. First record of the potentially invasive Pinctada imbricata fucata (Gould, 1850) (Bivalvia Pteriidae) along the Mediterranean coasts of Spain. Biodivers. J.12: 585-588. 10.31396/Biodiv.Jour.2021.12.3.585.588, Scuderi et al. 2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) are based only on morphological characters that we have shown to encompass the morphological variability of P. radiata. To date all the sequenced specimens of pearl oysters collected in the Mediterranean Sea are assigned to P. radiata, even from the same localities from where Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) identified specimens as P. fucata based on morphological characters. It is therefore highly likely that samples identified as P. fucata based on morphological characters by Cunningham Aparicio and Méndez (2021Cunningham AparicioB., MéndezI.2021. First record of the potentially invasive Pinctada imbricata fucata (Gould, 1850) (Bivalvia Pteriidae) along the Mediterranean coasts of Spain. Biodivers. J.12: 585-588. 10.31396/Biodiv.Jour.2021.12.3.585.588) also belong to P. radiata, suggesting that the latter species has reached the westernmost region of the Mediterranean Sea.
CONCLUSION
⌅This study clarifies the taxonomic identification and geographical distribution of pearl oysters in the Mediterranean Sea. Morphological characters proposed by Scuderi et al. (2019ScuderiD., BalistreriP., GermanàA.2019. Are Pinctada radiata (Leach, 1814) and Pinctada fucata (Gould, 1850) (Bivalvia Pteriidae) only synonyms or really different species? The case of some Mediterranean populations. Biodivers. J.10: 415-426. 10.31396/Biodiv.Jour.2019.10.4.415.426) as diagnostic for either P. fucata or P. radiata, based on which the presence of P. fucata was newly reported in the Mediterranean, were not validated by the integrative morphological-molecular approach carried out in this study. The earlier occurrences of P. radiata from the eastern and southern Mediterranean Sea are complemented with verified records in the western and northern Mediterranean, demonstrating that this non-native species is spreading further throughout the Mediterranean Sea. The integrative approach used in this study, combining molecular and morphological data, proved fruitful for the taxonomic identification of alien bivalves characterized by extensive variation in shell characters. This approach demonstrates once again that, in many organism groups, relying on only morphological characters might determine wrong taxonomic identification and mislead our estimates on the number and distribution of alien species. For these organisms, we strongly suggest requesting the verification of any new records of alien species with molecular data and recommend including in checklists only molecularly verified occurrences.