Mitochondrial DNA analysis reveals gene drift and structuring in the declining European piddock Pholas dactylus (L., 1758) confirming high vulnerability

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Highlights

  • Pholas dactylus is a historically valuable, and scarce, species in Europe where genetic data is lacking.

  • A differential pattern of genetic diversity was observed for P. dactylus in two areas of its European geographical distribution.

  • Data supports the existence of three different genetic units (Bay of Biscay, Villaviciosa and Black Sea).

  • Data from the studied areas reinforces the need for protection for this species and its habitats.

Abstract

Pholas dactylus is a historically valuable species with a relevant role in both environmental and biotechnological fields. It has become scarce in Europe due to habitat destruction and human overuse. This species is currently undergoing steep population declines, which have caused local extinction and/or distribution range contraction. Six different localities were sampled between the southern central region of the Bay of Biscay (Spain) and the Black Sea (Bulgaria and Romania) with the aim of describing for the first time its genetic variation patterns and assisting its conservation. Analyses using the mitochondrial Cytochrome Oxidase I gene revealed a high number of unique haplotypes in the Atlantic and Black Sea areas and significant genetic structuring (FST=0.15495 p<0.001, ΦST=0.36501 p<0.001). Significant differences were found between the regions since higher haplotype and nucleotide diversities were found in the Bay of Biscay (Dh=0.913, π=0.97%) than in the Black Sea (Dh=0.732, π=0.30%) and three different genetic units were discovered based on significant ΦCT values (western Bay of Biscay, Villaviciosa (the easternmost locality sampled within the Bay of Biscay) and the Black Sea) (ΦcT=0.41076 p<0.05). Globally, it seems that after different origins, gene drift has been acting on the species in its European geographical distribution. Results from this study reinforce the need for more efforts on obtaining data for this species and for a careful protection of its habitats.

Introduction

Pholas dactylus Linnaeus 1758, vernacularly known as the common piddock, is a bivalve mollusc belonging to order Myida, superfamily Pholadoidea, which comprises bivalve species with special adaptations for burrowing into soft rock or wood (Fig. 1). The Pholas genus presents extracellular luminescence and glows bluish-green in the dark; the luminescent glands lie in the syphons and mantle cavity, into which the luminous material is secreted (Wilbur and Yonge, 1968). In Ancient times, Pliny the Elder mentioned the luminescence in the mouths of people who ate Pholas and of such importance was this phenomenon that he even declared that the first king of Scotland had won his throne by consuming these clams (Bage, 1904).

P. dactylus occurs along the Eastern Atlantic coast from Norway in the north, going through the Iberian Peninsula and Morocco, to Cape Verde Islands in the south, as well as in the Mediterranean and Black Sea (Hill, 2006, Micu, 2007). Pholas drills its flask-shaped burrows in soft rocks (limestone, sandstone, chalk, calcarenite, shale, marl, clay) or even peat and waterlogged wood (Arias and Richter, 2012, Gil De Sola et al., 2012) located across the shore from the lowest intertidal (spring low tide) to the lower subtidal down to 10 m deep, large colonies being frequently found around 5 m deep. Due to its cryptic lifestyle, there is little knowledge about the species. The adult form is sessile and does not have dispersal potential, as it cannot leave the burrow nor can it re-burrow if dislodged, so recruitment by migration of adults is impossible (Pinn et al., 2005, Smith et al., 2011, Arias and Richter, 2012, Gil De Sola et al., 2012). The larvae are planktotrophic, with a larval stage of 45 days before the larva is competent to settle (Knight, 1984).

The drilling activity of P. dactylus engineers its own hard substrate habitat, causing an alteration of its physical and spatial structure and thus being considered as an ecosystem engineer species (Jones et al., 1994, Jones et al., 1997, Pinn et al., 2008). The concept of ecosystem engineering explains processes that involve species and their environment, are not directly trophic or competitive and result in the creation, maintenance, or modification of habitats. Ecosystem engineers can be autogenic, modifying the environment through their own physical biostructures (dead or living tissues) or allogenic, modifying the environment through their behaviour and activities (Commito and Rusignuolo, 2000, Norkko et al., 2006, Spooner and Vaughn, 2006), as is P. dactylus. Pholas activity creates a network of burrows and differentially accelerates erosion, increasing topographic complexity, modifying the availability and accessibility to different resources, which leads to an increase in species abundance and diversity within the habitat. Frequently, the galleries excavated by P. dactylus provide shelter to other species and/or their broods. In addition to its ecological importance, the species has an extensive record at the archaeological and cultural level (Lovell, 1884, Pinn et al., 2005, Gutierrez Zugasti, 2009), as well as possible applications as a bioindicator species and as source of the protein pholasin, which can be used as a probe of oxygen free radicals in living cells (Nourooz-Zadeh et al., 2006).

Historically, P. dactylus had a wide distribution range, but it has become rare in Europe recently due to the destruction and pollution of its habitat along with overexploitation by humans for food and fish bait (Michelson, 1978, Pinn et al., 2008, Arias and Richter, 2012). The species is highly protected nowadays and it is included in Annex II of the Convention on the Conservation of European Wildlife and Natural Habitats (Berne Convention) and in Annex II of the Protocol on Special Protection Areas and Biological Diversity of the Mediterranean of the Barcelona Convention (Ministerio de Medio Ambiente y Medio Rural y Marino, 2011).

In the Black Sea the most significant pressure has been chemical pollution and especially eutrophication as a result of nutrient enrichment (N, P and organic matter), most acutely experienced in 1970–1980s in the north-western Black Sea where there is high riverine input. P. dactylus cannot survive in anoxic or hypoxic conditions caused by eutrophication. Since the 1990s this pressure has been reduced due to tighter controls on pollution in the catchment of the Danube and other rivers which enter the north-western Black Sea. Whilst this pressure is now reduced, it is still posing a threat especially for non-EU countries surrounding the Black Sea, which are not bound by agreements like the Water Framework Directive (WFD). Basin-wide decline of the habitat at present is due to beam-trawling and coastal protection works, causing habitat destruction, smothering and siltation. It is recognized as rare and protected in Ukraine and Romania (Anistratenko, 1999, Micu, 2007), and although not mentioned in the Black Sea Red Data Book (Dumont, 1999) it was subsequently included in the inventory of aquatic and semi-aquatic Red List species, endangered in at least one country around the Black Sea in Annex 5 of the Black Sea Transboundary Analysis (Black Sea Economic Recovery Project, 2007). As ecosystem engineers, P. dactylus and Barnea candida create in the Black Sea a specific EUNIS level 5 habitat “A3.3 Infralittoral soft rock with Pholadidae”. This habitat type was assessed against the IUCN criteria as Endangered for the Black Sea region, within the project “Establishment of a European IUCN Red List of Habitats” (Gubbay et al., 2016). In the Bay of Biscay, again anthropogenic pressure has also been identified as the main cause of the Pholas disappearances (Arias and Richter, 2012). Despite this, a progressive increase of environmental protection at extended areas in Asturias as Sites of Community Importance (SCIs) and Special Protection Areas for birds (SPAs) under the Habitats (92/43/CE) and Birds (2009/147/CE) Directives have been taking place in the last years.

The main role of genetic approaches in the management and conservation of marine invertebrates is the identification of species and groups of individuals belonging to differentiated, disconnected genetic stocks, providing indirect measures of connectivity (Thorpe et al., 2000). Connectivity among populations shapes the genetic structure of species and determines the dynamics of metapopulation systems, how genetic diversity arises and is maintained within species, and the adaptability and resilience of populations to human pressures and environmental changes (Botsford et al., 2001), being crucial for an effective management of biological resources. Understanding the distribution of genetic variability is key for environmental resources management and conservation biology of marine species (Moritz, 1994, Palumbi, 2003, Cowen et al., 2006). Population connectivity plays a crucial role in local and metapopulation dynamics, genetic structure and population resilience, e.g., in response to human exploitation (Hastings and Harrison, 1994, Cowen et al., 2007, Weersing and Toonen, 2009, Puckett and Eggleston, 2012). Defining connectivity patterns for marine organisms is a challenging task since factors that affect connectivity (life history traits, habitat, hydrological regime, occurrence of geological/topographical boundaries, layout of coastline, etc.) act at very different geographic and temporal scales (Villamor et al., 2014). Most marine species release planktonic larvae which disperse over days up to months with the currents and thereby constitute the primary source of the dispersal capacity (Mileikowsky, 1971, Ward et al., 1994, Gilg and Hilbish, 2003). Direct labelling and tracking of larvae is seldom feasible, so genetic data are widely used for the indirect inference of population connectivity (Hellberg et al., 2002, Thorrold et al., 2002, Palumbi, 2003, Broquet and Petit, 2009, Cowen and Sponaugle, 2009, Lowe and Allendorf, 2010).

High levels of genetic differentiation have been often found in marine invertebrates, remarkably in corals and sponges, in which case may be related to their common biological characteristics like sessile life, great evolutionary age, limited ability to disperse and low homoeostatic capability (Solé-Cava and Thorpe, 1991). Previous research has shown that there is commonly an inverse relation between genetic connectivity among separated populations of a certain species and the extent to which said geographically separated populations have diverged (Burton and Feldman, 1982). It appears that species whose populations can maintain genetic exchanges via long-ranging propagules (eggs, seeds, planktotrophic larvae, adults) show little population differentiation over very large distances. Populations of species with low dispersal capacity, fragmented distribution and small stocks are much more vulnerable to overfishing or environmental changes (Thorpe et al., 2000). An additional difficulty is the high incidence of cryptic speciation in marine invertebrates, even in commercially important and comparatively well-studied species (Thorpe et al., 2000, Pogson, 2016).

The Cytochrome Oxidase subunit I (COI) gene has been very useful in population structure and phylogenetic studies since maternal inheritance, high copy number, relatively rapid mutation rate, and lack of recombination are all advantageous features for this type of studies (Palumbi, 2003). The COI gene has been used to properly identify species and therefore to reveal possible cases of cryptic speciation (Hebert et al., 2003, Szuster-ciesielska and Tustanowska-stachura, 2003, Plazzi and Passamonti, 2010, Jose and Mahadevan, 2016, Miralles et al., 2016). Moreover, COI has been demonstrated as a useful genetic marker to obtain information related to populations’ genetic structure in many marine invertebrates (Calderón and Turon, 2010, Campo et al., 2010, Muñoz Colmenero et al., 2015, Fourdrilis et al., 2016, Deli et al., 2017). It has been argued that to fully gauge haplotype variation at the species level, a strongly taxon-specific approach is necessary although typical sample sizes for molecular biodiversity assessment using DNA barcodes (COI) range from 5 to 10 individuals per species (Phillips et al., 2019). When working on endangered populations, the samples are usually difficult to obtain but genetic diversity, even if sample sizes are less than ideal, is still a relevant data (Pruett and Winker, 2008).

Processes related to dispersion, which ultimately determines patterns of connectivity, are highly linked to the biology and ecology of each species, but they are also contingent on the evolutionary history of the group and the geological history of the inhabited area. More taxon-specific analyses are therefore needed to better understand how dispersion and connectivity of marine species are shaped through time and geographic space, and their evolutionary and ecological consequences. Currently, there is not available genetic data about patterns of genetic variation in P. dactylus, although the species is already protected. It seems essential to provide more scientific support for the establishment of effective measures and conservation policies for the sustainable management of this species in its different distribution areas. In this work, we study the spatial genetic variation patterns for P. dactylus in two disjunct areas of its distribution range (Bay of Biscay and Black Sea) to characterize populations and to define units of management/conservation.

Section snippets

Sample collection and biometric analysis

Based on previous studies about the distribution of P. dactylus, four localities from the southern area of the Bay of Biscay and two from the Western Black Sea were selected: Tapia de Casariego, Zeluán, Peñarrubia and Villaviciosa in the Asturias coast (Perez, 2003, Arias and Richter, 2012) Costineşti in Romania (Micu, 2018) and Byala in Bulgaria (Gubbay et al., 2016) (Fig. 2). Samplings were authorized by competent authorities (i.e.: General Administrations of Maritime Fishing of the

Results

Thirty two samples were obtained from the southern central area of the Bay of Biscay (13 from Peñarrubia, 5 from Tapia, 7 from Villaviciosa and 7 from Zeluán) and twenty seven individuals were collected from the Black Sea region (15 from Costineşti and 12 from Byala (Fig. 2, Table 1)). The P. dactylus samples from Asturias cover a wide range of sizes (30.5–99.7 mm in length), the largest individuals being those from Zeluán (mean value = 82.4 ± 17.8 mm) and the smallest ones those from

Discussion

Useful information on past and recent demographics of a species and its populations can be inferred and interpreted from genetic data. Sea-level changes in the Pleistocene of Europe often led to the fragmentation of marine populations, creating a dynamic of spatial and demographic expansion and contraction over time (Provan and Bennett, 2008). In the last decades, cases of human-related range expansion have increasingly been reported (Rogers and Harpending, 1992, Grant and Bowen, 1998) and the

CRediT authorship contribution statement

Samuel López: Data was acquired, Data was analysed and interpreted, Writing - original draft, Take public responsibility for the content, Including participation in the concept, Design, Analysis, Writing - review & editing, Offering different perspectives on the results as to help in their discussion. Laura Miralles: Data was acquired, Data was analysed and interpreted, Writing - original draft, Take public responsibility for the content, Including participation in the concept, Design,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to thanks to the General Directorate of Marine Fisheries in the Principality of Asturias for providing us with sampling permits. Irene Fernandez (Zoology, UNIOVI) helped us with the Villaviciosa sampling.

Funding

This work was funded through a grant from the Asturias Government to Research Groups FC-GRUPIN-IDI/2018/000201. The present study is a contribution of the Biotechnology Institute of Asturias (IUBA) and Marine Observatory of Asturias (OMA).

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