A history of invasion: COI phylogeny of Manila clam Ruditapes philippinarum in Europe

Abstract

The Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) is now one of the top 5 most commercially valuable bivalve species worldwide. Originally from the Indo-Pacific region, it has been introduced in many countries for fisheries and aquaculture, including estuarine environments along Atlantic and Mediterranean European coasts. Yet despite its commercial value and widespread distribution, the precise origins of stocks remain speculative and the genetic diversity of introduced populations is poorly known. Thus, the aim of this work was to collect mtDNA COI (Cytochrome oxidase I) gene sequences from 5 European countries with Manila clam stocks and compare them with native Asian populations to evaluate their genetic diversity and identify possible routes of invasion. The COI gene sequencing supported a strong founder effect in the European populations with 3 main haplotypes occurring at high frequencies, derived from Japan. However, high haplotype diversity was also observed due to the occurrence of 10 rare haplotypes. This supports hypotheses (i) there have been additional, previous unrecorded, introductions as previously hypothesized by analysis of 16S rDNA, and (ii) there has been a limited loss of genetic diversity in introduced populations, as previously suggested by microsatellite data. This is the first genetic comparison of Manila clam populations introduced in to Europe with native clams. Genetic data herein presented are fundamentally important for the traceability of clam products and stock management programmes and will also inform discussion on the potential resilience of exploited Manila clam populations.

Introduction

Among commercially exploited bivalves, the Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) is of considerable international importance and considered among the top 5 most commercially valuable bivalve species worldwide (over 250,000 tons for year) (Astorga, 2014). Originally distributed in the Indo-Pacific region it has been introduced in many countries for fisheries and aquaculture (Gosling, 2003), including European Atlantic and Mediterranean coastal waters (Gosling, 2003). As reported by Flassch and Leborgne (1992), until the 1990s the main European stocks originated from a small pool of organisms introduced from North America (see Table 1 for a summary of initial introductions in Europe).

Following the available data on licensed introductions, the first introductions in Europe dates back to 1972–1974 in Arcachon Bay, France by IFREMER (Institut Français de Recherche pour l’Exploitation de la Mer). Flassch and Leborgne (1992) reported that a total of 500,000 spat, and 1000 adults from Puget Sound (South Western Canada, Pacific coast) were introduced into the Arcachon Bay, roughly representing a total biomass of 70 kg. The same population from Puget Sound was used for the first introduction of Manila clam in the UK in 1980, at the MAFF (Ministry of Agriculture, Fisheries and Food) Fisheries Laboratory, Conwy-North Wales (Humphreys et al., 2015). The near-by Menai Strait was identified as the location of the first introduction into UK coastal waters in 1983 (Humphreys et al., 2015). In the same year, the first introduction in the Northern Adriatic Sea also occurred, conducted by the Co.S.PA.V (Consorzio per lo Sviluppo della Pesca e dell’Acquacoltura del Veneto) in the Venice lagoon using seed from Great Britain (Breber, 1985). In a short period of time Manila clam was introduced in other Adriatic coastal lagoons, namely Marano, Caleri, Scardovari, Goro (Pellizzato, 1990). All these first introductions were conducted with clams coming from SeaSalter Shellfish Company (M. Pellizzato, pers. Comm.) which operated from hatcheries in south-east and north-west England, and the company was established with clams from Conwy (Humphreys et al., 2015). In Spain, Manila clam was already occurring in the mid ‘80s (Perez-Camacho and Cuna, 1985) in many different coastal areas (Galicia, Cantabria, Andalusia, and Cataluña). The first report of Manila clam in Portugal dates back to 1984 in Ria Formosa (Algarve) probably originated from Spain (Ruano and Sobral, 2000), even if no information about the status of the Spanish “source” population (hatchery or naturalised) is available. The species is not yet licensed in Portugal (Chainho et al., 2015) even if aquaculture was the most likely vector of introduction (Chainho et al., 2015). However, since the ‘80s, naturalised Manila clam populations have been reported in many estuarine systems all over the country (Gaspar, 2010, Chainho, 2014, Chainho et al., 2015, Velez et al., 2015a, Velez et al., 2015b). Today Manila clam is considered the dominant bivalve species in the Tagus estuary and is one of the most abundant clams in the Ria de Aveiro and Sado estuary (Chainho, 2014, Velez et al., 2015a).

Nowadays, the production of Manila clam in Europe derives mainly from fisheries of naturalised populations, established after human-mediated introductions. This is the case in France, specifically Arcachon Bay, where the whole production derives from the original introduced and naturalised population (Bald et al., 2009, Sanchez et al., 2014), and also England (Humphreys et al., 2015), Spain (e.g. the Bay of Santander- Bidegain and Juanes, 2013) and Portugal (Chainho et al., 2015). Detailed literature data are available for UK, where the first reported naturalised Manila clam population was observed in Poole Harbour (Jensen et al., 2004), where the first licensed introduction dates back to 1988 from seeds originated from Conwy hatchery, and wild clams appeared about two years latter (Humphreys et al., 2015). Between 1980 and 2010 the Manila clam has become naturalised in 11 British estuaries. The most extensive newly established wild populations is in Southampton Water, which lies about 48 km east of Poole Harbour, and where Manila clam likely arrived in 2002 (Humphreys et al., 2015). It is possible that this has originated via natural larval dispersal from Poole Harbour (Herbert et al., 2012) or anthropogenic means (Humphreys et al., 2015).

Aquaculture facilities have been also successfully established for Manila clam in UK, Italy (Northern Adriatic Sea) and in Spain, especially in Galicia (Robert et al., 2013). In Spain, hatcheries mainly provide seeds for local associations of producers. Most of the production takes place in private parks (concessions for a period of years) and on beaches that are managed by local associations (Robert et al., 2013). In Italy, Manila clam spread occurred rapidly, and quickly populations became naturalised (Pellizzato, 1990) thus its exploitation became the most economically important fishing activity, especially in the Venice Lagoon (see Boscolo Brusà et al., 2013 for a complete list of references). However, due to the initial lack of reliable regulation and unsustainable exploitation of fisheries resources, there has been a constant decrease in clam production (Boscolo Brusà et al., 2013), which determinated a recent transition from clam fishing to clam farming activities, and to the rational management of natural spat (Boscolo Brusà et al., 2013). Currently, in the Venice lagoon most clam harvesting is carried out in licensed areas directly managed by farmers (Boscolo Brusà et al., 2013), using seeds derived from natural spat. This system has been already established in other Northern Adriatic Sea lagoons, like Goro lagoon, where the production remained stable for almost 3 decades (Bartoli et al., in press). In general, the problem for Manila clam cultivation in Europe is the same as global shellfish aquaculture: high quality seed availability (Robert et al., 2013). Although efforts have been made to improve the hatchery production, clam farming of Manila clam still depends on natural seeds (Robert et al., 2013).

As underlined in previous paragraphs, Manila clam is a valuable economic resource for some European countries. However, as pointed out by Astorga (2014), although aspects of the species biology have been studied genetic resources are still largely unknown. For several fisheries and aquaculture commercial species, especially fish, biotechnology and genetic research have developed significantly in the last decade (Astorga, 2014); however similar applications for valuable molluscs have been minimal (Astorga, 2008, Astorga, 2014) and for Manila clam in particular. In fact, whole genome reference sequences, high-density SNP genotyping arrays or genotyping-by-sequencing have been developed especially for fish (Yáñez et al., 2015). As for Manila clam, few studies have been devoted to the genetic diversity and structure of populations in its native range (see as examples Sekine et al., 2006, Vargas et al., 2008, Liu et al., 2007, Mao et al., 2011, An et al., 2012, Kitada et al., 2013, Nie et al., 2015) and in introduced ecosystems (Chiesa et al., 2011, Chiesa et al., 2014, Chiesa et al., 2016a, Mura et al., 2012, Hurtado et al., 2011). Yet a comparative study of native and introduced populations has not previously been undertaken and no genetic information is available concerning the differences occurring among productive stocks worldwide, or potential invasion pathways that might compromise the ability to perform predictions of genetic diversity and population structure of non-indigenous taxa (Holland, 2000).

The genetic structure of an invasive population depends on several factors, including the effective population size at the time of introduction and the genetic diversity of the source population (Holland, 2000). If an introduction occurs as a single event, starting from a limited number of founders, population genetic theory predicts that alleles will be fixed and lost at an accelerated rate relative to the source population (Mayr, 1963, Hartl and Clark, 1997, Holland, 2000). The gene pool of the introduced population is expected to be limited, as a result of the stochastic process of the introduction mechanism (Holland, 2000). However, if the introduction involves a large genetically diverse assortment of individuals, it is expected to have little or no reduction in heterozygosity and allelic diversity relative to the gene pool of the source population (Holland, 2000). In fact, a founding population which derives from numerous previously isolated populations has the potential to produce a genetically highly diverse assortment of offspring. It has been already proposed by Roman and Darling (2007) that invasions from multiple discrete source populations, or admixture, may be the standard rather than the exception in invasion biology and that the co-occurrence of mitochondrial lineages, geographically separated in the native range, could be considered an evidence of multiple introductions events (Taylor and Keller, 2007).

Furthermore, genetic data on Manila clams is fundamental for studies associated with clam traceability and safety, preventing fraud and supporting management programmes of exploited populations. This is particularly important for a highly exploited resource like Manila clam, both for fisheries and aquaculture. In fact, the erosion of the genetic diversity determinates a high risk of introgression and a reduction of fitness of the exploited populations, and also their resilience capability as relict populations (Frankham et al., 2009). In the Venice lagoon, an overexploitation of Manila clam that has occurred in the last decades has resulted in a huge reduction of the naturalised population (Boscolo Brusà et al., 2013) with possible consequences for genetic diversity and demographic structure.

Previously, studies conducted on Manila clam populations from the Northern Adriatic Sea, Portugal (Ria de Aveiro) and Spain (Galician coast) demonstrated a strong founder effect by 16SrDNA gene sequencing, but also enhanced haplotype diversity occurring in introduced populations (Chiesa et al., 2011, Chiesa et al., 2014). Moreover, microsatellite genotyping in the same populations showed a limited loss of genetic diversity, and even though several loci were affected by null alleles, globally the number of alleles was comparable to those observed in native Asian populations (Chiesa et al., 2011, 2016a, in press).

Considering that previous studies on Asian populations were also conducted with COI gene fragment sequencing (Sekine et al., 2006, Mao et al., 2011, Kitada et al., 2013), the present work aimed to collect mtDNA COI gene sequences also from 5 European countries hosting Manila clam aquaculture and fishing activities, and for the first time to compare genetic diversity between these introduced stocks and native Asian populations. This is the first genetic study to investigate invasion routes of Manila clams in Europe and the genetic diversity of commercial stocks, which will contribute to the basic knowledge in the field of invasion biology, and support management programmes of this valuable economic resource in European countries.