Genetic diversity of introduced Manila clam Ruditapes philippinarum populations inferred by 16S rDNA

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Highlights

  • Ruditapes (Venerupis) philippinarum is a valuable economic seafood.

  • Genetic structure of invasive populations was never investigated in Europe.

  • 16S rDNA was investigated in 12 introduced Mediterranean and Atlantic populations.

  • Occurrence of 11 haplotypes were detected.

  • Multiple introduction events from different recruitment stocks occurred.

Abstract

Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) – is one of the most successful marine invaders, being introduced worldwide for aquaculture and fisheries. Genetic diversity and structure of its populations are largely unknown, especially in the invaded environments. Herein we present molecular genetic data on 12 introduced Manila clam populations in Italy, Spain, and Portugal from both Mediterranean (Adriatic) and Atlantic sampling sites. The phylogenetic information was investigated by the direct sequencing of 16S mitochondrial DNA. Results of mtDNA analyses showed the occurrence of 11 haplotypes for European introduced populations, as a consequence of multiple founder effects from different source populations. Rp hap1 was the most frequent, shared among all populations. The other 10 haplotypes were rare and distributed at local scale, in agreement with what was observed in other invasive bivalves. Biogeographic and phylogenetic analyses based on 16S rDNA of introduced Manila clam populations showed a complex scenario, dominated by multiple pools of individuals coming from different sources, as a consequence of multiple introductions.

Introduction

Among the variety of worldwide commercial seafood products, cultivation of molluscs, mainly bivalves, has a long history and it has become largely established in many countries (Bostock et al., 2010). Moreover, many bivalves species have been introduced outside their native habitats for aquaculture and fisheries purpose (Grigorakis and Rigos, 2011); they became successful invaders, occurring at high densities and accounting for the major proportion of the benthic faunal biomass (Sousa et al., 2009). In particular, species belonging to the family Veneridae (Rafinesque, 1815) are actually exploited for human consumption (Edwards, 2005), mostly due to the fishing and farming of Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850).

R. philippinarum represents itself almost 20% of worldwide mollusc total production (Edwards, 2005), being one of the most successful invaders (FAO, 2013). Originally from the Indo-Pacific region of Japan, Korea and China (Gosling, 2003), it was introduced into the west coast of North America, Atlantic (Portugal, France, Spain, Ireland, England) and Mediterranean European coasts (France, Italy) (Gosling, 2003). Moreover, aquaculture trials resulted in seed being imported into French Polynesia, the US Virgin Islands, Norway, Germany, Belgium, Tunisia, Morocco, and Israel (FAO, 2013). In Europe, the species was introduced for commercial cultivation (Bald et al., 2009); since its introduction, production of Manila clam improved, and Europe became the leading area in the world after China (Guo et al., 1999).

Although commercially exploited and deeply manipulated by human activities, Manila clam is still poorly known under many biological points of view, especially genetics. Surprisingly, few papers have been published concerning genetic diversity of native and introduced populations of Manila clam. In China, Korea and Japan, less than a dozen of manuscripts have been published using allozyme markers (see Vargas et al., 2008), mtDNA sequencing (see Kitada et al., 2013), AFLP (Liu et al., 2007); RAPDs and microsatellites (see An et al., 2012, Kitada et al., 2013). In Europe, we can actually account only two papers on Italian populations (Chiesa et al., 2011, Mura et al., 2012).

Genetic data in invasion biology are fundamental from many points of view (- Frankham et al., 2009, Estoup and Guillmaud, 2010): to correctly identify an invasive species (Scalici et al., 2009); to understand whether or not the introduced species could become invasive, to construct predictive models of future spread; to evaluate the evolutionary changes in invasive species and their impact on native species.

Moreover, molecular genetics could provide valuable data for the tracing and tracking of seafood products, as already requested by the European Union law EC No. 2065/2001 (Filonzi et al., 2010, Caldelli et al., 2014), on the basis of their geographic distribution. The identification of informative genetic markers and their application to Manila clam populations is particularly important for the tracing of harvested clams. Clams cultivation and fisheries in both Mediterranean and Atlantic coastal lagoons frequently occurs in low or moderated polluted areas (Sfriso et al., 2008, Freitas et al., 2012) posing risks for human's safety, especially in coastal communities. The genetic profiles of cultivated and harvested bivalves populations can certify their geographic origin, to guarantee consumer's safety and product's quality control. These informations will improve the value of bivalves products, both for consumers and producers.

We herein present data on 12 introduced Manila clam productive populations belonging to Italy, Spain, and Portugal to assess their genetic diversity on a larger scale, on both Mediterranean and Iberian Atlantic populations. We investigated the biogeographic and the phylogenetic information throught the direct sequencing of 16S mitochondrial DNA, which has been proven to be a useful marker for intraspecific diversity in invasive bivalves (Stepien et al., 1999), and in Veneridae family (Canapa et al., 2003, Kappner, 2006, Mikkelsen et al., 2006).

Section snippets

Sampling procedures

Twenty adult individuals of Manila clam were collected from 12 introduced harvested populations from both Mediterranean and Atlantic coasts of Europe. Sampling sites were distributed in Northern Adriatic Sea from the Marano Lagoon (two sites), the Venice Lagoon (three sites) to the River Po Delta (two sites) and Sacca Scardovari (one site). Moreover, one population was collected in North Western Spain (Galician coast) and three from Northen Portugal (Ria de Aveiro Lagoon) (Fig. 1 and Table 1).

DNA extraction and purification

Results

The 16S rDNA sequences were successfully sequenced for 133 individuals of R. philippinarum (see Table 1) and two of R. decussatus and were aligned unambiguously for 438 bp or 407 bp depending on the species. The final dataset comprised 146 sequences considering also those downloaded from Genbank.

The overall number of mutations among aligned sequences of R. philippinarum (137) was 12, considering both original samples (133) and reference ones (4). The mutations were identified at positions 23,

Discussion

The results herein presented describe for the first time the genetic diversity of introduced Manila clam populations in Europe, observing twelve haplotypes in 12 introduced populations analyzed by the direct sequencing of 16S rDNA. The European introduced populations were characterized by one common haplotype, Rphap1, whilst the other 10 haplotypes were represented by only 1 or 2 sequences. This scenario was already observed in other species of invasive molluscs, like Mytilopsis sallei (Recluz,

Acknowledgements

Authors would like to thank G.R.A.L. (Gestione Risorse Alieutiche Lagunari) of Venice Lagoon for Venice Samples and Dr. Immaculada Llorents Gumbau for collection of Spanish samples. The research was funded by own funds of Molecular Sciences and Nanosystems Dept. (Venice) and Life Sciences Dept. (Parma). Moreover, this work was supported by the Spin off “Gen Tech” of the University of Parma, the European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT)

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