Project
title / Population genetics of shellfish in British waters
/ DEFRA
project code / MF0226

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
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Project title / Population genetics of shellfish in British waters
DEFRA project code / MF0226
Contractor organisation and location / Royal Holloway University of London, School of Biological Sciences, Egham, TW20 0EX
(Subcontractor - University of Hull, Department of Biological Sciences, Hull, HU6 7RX)
Total DEFRA project costs / £ 253,246 (budget)
Project start date / 01/08/00 / Project end date / 30/09/03
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 4

Project
title / Population genetics of shellfish in British waters
/ DEFRA
project code / MF0226

Scientifically sound management of exploited shellfish resources relies on basic knowledge of the biology of species, including information on population structure and recruitment patterns. Such information influences the development and execution of management strategies, the design of biological sampling programmes, and strategies for conserving biodiversity. Little is known of the genetic population structure of commercially exploited shellfish species in British waters, resulting in considerable uncertainties about the interdependence of exploitation in the different fishing areas and about recruitment predictions underlying management of the resource. The proposed research project aimed to develop and employ genetic markers to investigate the extent of population structuring in four selected shellfish species of commercial importance (Edible Crab Cancer pagurus, Common cockle Cerastoderma edule, Brown shrimp Crangon crangon and Common Whelk Buccinum undatum). These species were chosen to represent a range of population distributions, life histories and migratory behaviour, with the aim that general findings would therefore allow more accurate predictions of population structure in other species.

There are two major components to the present project: a methodological aspect involving development of protocols for identification of suitable DNA regions to be used as genetic markers; and a population genetc analysis aspect. The latter involves use of the genetic markers developed to screen geographical samples, providing population genetic information as a framework for improved understanding of the biology and stock structuring of the target shellfish species. Both components were pursued in three of the four species (Edible Crab, Common Cockle, Common Whelk), but unusual genomic characteristics of the Brown shrimp meant that the population screening had to be abandoned for this species. Substantial progress has been achieved in both methodological development and data production, with all original proposal objectives being achieved to some degree.

Sampling. With help from CEFAS staff, large samples (50-100 individuals per site) were collected for all four species from locations around the UK, covering major fishery areas, as well as in continental Europe to assess links between UK and non-UK fisheries. Samples collected over years and repeated samples within areas allow testing for temporal and micro-geographical variation.

Marker development. Difficulties occurred with routine extraction and storage of DNA: under standard storage conditions DNA degraded rapidly. Two alternatives were developed for reliable use of DNA-based markers in shellfish species: a novel “hot-shot” method of DNA extraction (boiling in NaOH); CTAB extraction and immediate transfer to PCR amplification steps (i.e. no storage). We advise adoption of such protocols for DNA studies of shellfish, especially molluscs.

Isolation of microsatellite DNA regions suitable for marker development was more difficult and time consuming in the target species than in other taxa (e.g. fish, cephalopods, birds) of which we have considerable experience. Substantial effort put into isolation did however generate information regarding the nature of genomes of the target species: microsatellites are not uncommon, but predominantly are very long, complex in nature, and appear to occur within highly repetitive DNA regions. Whilst this was a complication for the current project, it provides valuable direction for future studies of these and other shellfish species. Successful isolation of marker loci was achieved for 3 species: whelk, cockle and crab. The total absence of suitable DNA regions in shrimp resulted in abandoning this approach, and an alternative method was tested: AFLP markers. AFLP development corroborated the pattern in shrimp of a genome of highly repetitive nature, making it refractory to useful marker development, although very preliminary results do suggest that AFLP may be a useful approach for examining population differentiation in other species.

Population genetic analysis. Samples of three target species (crab, cockle, whelk) from populations all around UK coasts have been screened for genetic variation. High levels of genetic polymorphism exist within all marker loci, and as sample sizes were purposefully high (60-100 individuals per site) the markers allow powerful tests for differentiation. Initial analysis indicates:

Common Whelk (Buccinum undatum). Global tests of the component of genetic variation distributed among sites (FST = 0.034) indicate significant structuring in the UK whelk population. Pairwise tests indicate relatively low, but significant, genetic differences between almost all samples. There is some indication of hierarchical genetic structure with local populations less genetically different than more distant ones, although there is no absolute correlation of genetic differentiation with geographical distances between sites (i.e. no isolation by distance) or with any obvious geographical or hydrographic features. It therefore appears that there is no extensive genetic mixing (gene flow = effective migration) between populations, even for sites 10km apart, and there might be “mosaic” (i.e. unstructured) genetic differentiation of this species around the UK.

Common Cockle (Cerastoderma edule). Global FST (=0.020) tests again indicate significant structuring in the UK cockle population. Pairwise tests indicate that samples within localised regions (e.g. Burry Inlet – Carmarthen) are not significantly different (i.e. they represent a single population), and that low, but significant genetic differences occur between wider geographical regions. No distinct geographical pattern is obvious, although west, south and east coast samples appeared to be separated. It appears that short-term extensive genetic exchange is not occurring between cockle populations around UK coasts.

Edible crab (Cancer pagurus). Global tests of the component of genetic variation distributed among sites (FST = 0.013) indicate significant structuring in the UK crab population. Pairwise tests indicate relatively low, but significant, genetic differences between almost all samples. There is no correlation of genetic differentiation with geographical distances between sites (i.e. no isolation by distance) or with any obvious geographical or hydrographic features (although the geographical scale of sampling was not suited to rigorous tests of the effect of very localised features). As with whelk, results indicate a mosaic population structure, suggesting that local populations have little effective genetic exchange with other areas.

In all three species, tests between temporally separate samples from the same site indicated no significant allele frequency differences, suggesting that local populations are relatively stable over time. Tests between microgeographic samples (several sites within a locality) indicated no allele frequency differences in most cases, indicating that effective genetic exchange does occur between local populations (over no more than several 10s of km) in all three species.

Conclusions. Some general conclusions can be drawn on population genetic processes in these species, although further, more locally targeted sampling would be required to confirm details. Uniformly high levels of genetic variation across all samples in all species suggests that local populations have not been subject to recent population size bottlenecks or high inbreeding, suggesting that local effective population sizes (responsible for determining genetic properties of populations) may be large and stable. Low but significant levels of genetic differentiation between almost all sites, of a geographically unstructured nature, indicates that effective genetic exchange (i.e. migration / dispersal) is not occurring between populations even over relatively small distances (10s of km), at least in the short term. Overall values of the component of between-population variation (FST) are higher in whelk (0.034) than cockle (0.020), and both are higher than in crab (0.013), in line with predictions on relative levels of dispersal drawn from species biology: although gene flow is lower than expected from the life history characteristics (at least for cockle and crab), general predictions on relative rates of dispersal may still be applicable.

Recommendations. Our results show significant genetic differences at surprisingly small scales in all three species tested, and therefore provide good evidence for limited demographic connectivity among the shellfish populations surveyed here. It is likely that essentially self-recruiting populations may show demographic isolation on an even smaller geographic scale. Significant genetic differences among fishing areas indicate very limited dispersal and demographic independence of different stocks, which should therefore be managed independently. Local depletion is unlikely to be compensated for by immigration in the short term, and will therefore depend on local population recovery and recruitment. Clearly, the exact definition of management units will require additional genetic and demographic data, and will also depend on the feasibility of data collection, analysis and management implementation. Locally differentiated stocks with limited exchange may differ in vital demographic parameters. Furthermore, the existence of locally differentiated populations suggests that excessive local depletion may result in a loss of genetic diversity and any associated locally adaptive attributes, and should therefore be avoided as part of biodiversity conservation efforts.

The results of our project therefore have relevance to DEFRA policy issues, in particular in the definition of appropriate management units in these species. Because of the continued decline in finfish stocks such as cod, haddock and herring, shellfish play an increasingly important role for the UK fisheries industry - detailed knowledge of population structure and recruitment dynamics will be essential for conservation and sustainable exploitation of this resource.

CSG 15 (Rev. 6/02) 4

Project
title / Population genetics of shellfish in British waters
/ DEFRA
project code / MF0226
Scientific report (maximum 20 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.

CSG 15 (Rev. 6/02) 4

Project
title / Population genetics of shellfish in British waters
/ DEFRA
project code / MF0226

1. Introduction

The idea that fisheries management should be based on local self-sustaining populations rather than the typological species can be traced back to the turn of the 19th century (Heincke 1898, Hjort 1914). Fisheries models developed since then are based on ‘stocks’, which are the unit for the estimation of fisheries parameters such as the number of spawning individuals and recruitment (Hilborn & Walters 1992). Despite this long history of the stock concept (Sinclair 1998, Carvalho & Hauser 1994), our knowledge of the population structure of many marine species is still very limited, not only because of the apparent lack of environmental barriers to dispersal, but also because of the ecology and life history of many species, which seems to promote large scale dispersal (Hauser & Ward 1998). Furthermore, early molecular studies based on protein variability often revealed little genetic differentiation, suggesting the existence of large panoceanic populations. However, recently the combination of improved sampling design and more sensitive genetic markers has provided evidence for population structure on a surprisingly small scale (e.g. cod, Hutchinson et al. 2001, Ruzzante et al. 2000), suggesting limited larval dispersal and effective local retention (Palumbi 2003).

Recent evidence from larval biology suggests that long distance dispersal, although important evolutionarily (Strathmann 1978, Duda & Palumbi 1999), may be rare (Palumbi 2001), and that most of the recruitment in demersal fish and benthic invertebrates may stem from local sources. Oceanographic features such as currents or eddies play an important role in dispersal and retention of pelagic larvae, and may also strongly affect larval mortality (Bailey et al. 1997). Furthermore, behavioural mechanisms, such as vertical migrations exploiting currents at different depths, can greatly influence the direction and extent of horizontal advection (Bilton et al. 2002). Evidence for localized self-recruitment comes from unexpected genetic subdivisions in marine species (Avise 1992, Taylor & Hellberg 2003), the persistence of demersal fish with pelagic larvae on isolated oceanic islands (Hourigan & Reese 1987, White 1998) and information on larval distribution (Bailey et al. 1997, Hay & McCarter 1997) and behaviour (Bilton et al. 2002). It has thus become clear that the potential dispersal suggested from larval duration is often not realized, and that population genetic studies are valuable and necessary even in species with long-lived pelagic larval stages.

Molecular genetic methods can be extremely powerful tools in fisheries management by enabling the identification of relatively isolated populations that will react independently to exploitation and may differ in the parameters used in fishery models (e.g. growth, mortality, recruitment) (Carvalho & Hauser 1994). In particular, microsatellite DNA markers are being increasingly used in marine species, where they have revealed population differentiation that other markers have not (e.g. Shaw et al. 1999). Microsatellites consist of 1-5 base pair (bp) repeats that form tandem arrays up to 300 bp in length, and exhibit high levels of allelic variation in repeat number. Polymorphism exhibited by specific microsatellites is readily detected by amplification of the microsatellite through the use of oligonucleotide primers specific to the non-repetitive regions that flank the repeat array, in combination with the polymerase chain reaction (PCR). Allelic variation is detected by gel electrophoresis of the PCR products, commonly on automated DNA sequencer systems. However, species-specific primers usually need to be isolated, incurring considerable costs and effort. Despite the development effort usually required for the isolation of species specific primers, microsatellites are at present the method of choice for many population genetic applications, especially where genetic differentiation may be limited, as in geographically proximate populations or in species with high potential migration rates (Wright & Bentzen 1994).